Anti-cancer compositions and methods

ABSTRACT

Compositions and pharmaceutical compositions including one or more selenium-containing COX-2 inhibitors are provided according to aspects of the present invention. Methods of treating a subject having or suspected of having cancer are provided according to aspects of the present invention which include administering a therapeutically effective amount of a pharmaceutical composition including a selenium-containing COX-2 inhibitor.

REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional PatentApplication Ser. No. 61/759,034, filed Jan. 31, 2013, the entire contentof which is incorporated herein by reference.

GOVERNMENT SPONSORSHIP

The invention was made with government support under Grant Nos. CA127892and CA136667 awarded by The National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to anti-cancer compositions andmethods. In specific aspects, the present invention relates tocompositions including one or more selenium-containing cyclooxygenase-2(COX-2) inhibitor compounds, methods for treatment and/or prevention ofpathological conditions in a subject using one or moreselenium-containing COX-2 inhibitor compounds and methods for synthesisof particular selenium-containing COX-2 inhibitor compounds.

BACKGROUND OF THE INVENTION

In spite of recent medical progress, cancer continues to be one of themost common and deadly diseases. Elucidation of biochemical pathwaysinvolved in development and progression of various cancers is importantto identify potential anti-cancer treatments as well as to developagents effective to regulate such pathways in other aspects of healthand disease.

Compositions and methods are required to inhibit cancer cell survivaland proliferation. In particular, compositions and methods are requiredto inhibit COX-2 activity in cancer cells, reduce survival and/or reduceproliferation of cancer cells.

SUMMARY OF THE INVENTION

Compositions including one or more selenium-containing COX-2 inhibitorsare provided according to aspects of the present invention.

Compositions including a selenium-containing COX-2 inhibitor areprovided according to aspects of the present invention, wherein theselenium-containing COX-2 inhibitor is selenocoxib-1-glutathione(selenocoxib-1-GSH).

Compositions including a selenium-containing COX-2 inhibitor areprovided according to aspects of the present invention, wherein theselenium-containing COX-2 inhibitor is selenocoxib-1-cysteine.

Compositions including a selenium-containing COX-2 inhibitor areprovided according to aspects of the present invention, wherein theselenium-containing COX-2 inhibitor is selenocoxib-1-N-aceylcysteine(selenocoxib-1-NAC).

Pharmaceutical compositions including one or more selenium-containingCOX-2 inhibitors and a pharmaceutically acceptable carrier are providedaccording to aspects of the present invention.

Pharmaceutical compositions including: a pharmaceutically acceptablecarrier; and one or more of: selenocoxib-1-GSH, selenocoxib-1-cysteineand selenocoxib-1-NAC, are provided according to aspects of the presentinvention.

Pharmaceutical compositions including one or more selenium-containingCOX-2 inhibitors and a pharmaceutically acceptable carrier are providedaccording to aspects of the present invention, wherein thepharmaceutical composition is formulated for topical application.

Pharmaceutical compositions including: a pharmaceutically acceptablecarrier; and one or more of: selenocoxib-1-GSH, selenocoxib-1-cysteineand selenocoxib-1-NAC, are provided according to aspects of the presentinvention, wherein the pharmaceutical composition is formulated fortopical application.

Pharmaceutical compositions including one or more selenium-containingCOX-2 inhibitors and a pharmaceutically acceptable carrier are providedaccording to aspects of the present invention, wherein thepharmaceutically acceptable carrier includes a particulate carrier.

Pharmaceutical compositions including: a pharmaceutically acceptablecarrier; and one or more of: selenocoxib-1-GSH, selenocoxib-1-cysteineand selenocoxib-1-NAC, are provided according to aspects of the presentinvention, wherein the pharmaceutically acceptable carrier includes aparticulate carrier.

Pharmaceutical compositions including one or more selenium-containingCOX-2 inhibitors and a pharmaceutically acceptable carrier are providedaccording to aspects of the present invention, wherein thepharmaceutically acceptable carrier includes a nanoparticulate carrier.

Pharmaceutical compositions including: a pharmaceutically acceptablecarrier; and one or more of: selenocoxib-1-GSH, selenocoxib-1-cysteineand selenocoxib-1-NAC, are provided according to aspects of the presentinvention, wherein the pharmaceutically acceptable carrier includes ananoparticulate carrier.

Pharmaceutical compositions including a plurality of nanoliposomes areprovided according to aspects of the present invention, wherein thenanoliposomes include selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, and wherein the nanoliposomeshave an average particle size in the range of 1-100 nm.

Pharmaceutical compositions including one or more selenium-containingCOX-2 inhibitors, one or more additional therapeutic agents, and apharmaceutically acceptable carrier are provided according to aspects ofthe present invention.

Pharmaceutical compositions including: a pharmaceutically acceptablecarrier; one or more of: selenocoxib-1-GSH, selenocoxib-1-cysteine andselenocoxib-1-NAC; and one or more additional therapeutic agents, areprovided according to aspects of the present invention.

Methods of treating a subject are provided according to aspects of thepresent invention which include administering a therapeuticallyeffective amount of a pharmaceutical composition including aselenium-containing COX-2 inhibitor. The subject is a human according toaspects of the present invention. Optionally, the pharmaceuticalcomposition is formulated as a topical composition. In a further option,the pharmaceutical composition includes a particulate or nanoparticulatepharmaceutically acceptable carrier. In another option, thepharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition.

According to aspects of the present invention, methods of treating asubject include administering a therapeutically effective amount of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof. Thesubject is a human according to aspects of the present invention.Optionally, the pharmaceutical composition is formulated as a topicalcomposition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

Methods of treating a subject having or suspected of having cancer areprovided according to aspects of the present invention which includeadministering a therapeutically effective amount of a pharmaceuticalcomposition including a selenium-containing COX-2 inhibitor. The subjectis a human according to aspects of the present invention. Optionally,the pharmaceutical composition is formulated as a topical composition.In a further option, the pharmaceutical composition includes aparticulate or nanoparticulate pharmaceutically acceptable carrier. Inanother option, the pharmaceutical composition includes nanoliposomeshaving an average particle size in the range of 1-100 nm. In a furtheroption, an additional therapeutic agent is included in thepharmaceutical composition.

According to aspects of the present invention, methods of treating asubject having or suspected of having cancer include administering atherapeutically effective amount of a pharmaceutical compositionincluding selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. The subject is a humanaccording to aspects of the present invention. Optionally, thepharmaceutical composition is formulated as a topical composition. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition.

Methods of treating a subject having or suspected of having breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma are provided according to aspects of the present inventionwhich include administering a therapeutically effective amount of apharmaceutical composition including a selenium-containing COX-2inhibitor. The subject is a human according to aspects of the presentinvention. Optionally, the pharmaceutical composition is formulated as atopical composition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

According to aspects of the present invention, methods of treating asubject having or suspected of having breast cancer, lung cancer,prostate cancer, colon cancer, liver cancer or melanoma includeadministering a therapeutically effective amount of a pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. The subject is a humanaccording to aspects of the present invention. Optionally, thepharmaceutical composition is formulated as a topical composition. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition.

Methods of treating a subject wherein the subject has or is suspected ofhaving cancer characterized by increased COX-2 protein or nucleic acid,increased COX-2 activity and/or increased Akt activity compared to acontrol are provided according to aspects of the present invention whichinclude administering a therapeutically effective amount of apharmaceutical composition including a selenium-containing COX-2inhibitor. The subject is a human according to aspects of the presentinvention. Optionally, the pharmaceutical composition is formulated as atopical composition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

According to aspects of the present invention, methods of treating asubject wherein the subject has or is suspected of having cancercharacterized by increased COX-2 protein or nucleic acid, increasedCOX-2 activity and/or increased Akt activity compared to a controlinclude administering a therapeutically effective amount of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof. Thesubject is a human according to aspects of the present invention.Optionally, the pharmaceutical composition is formulated as a topicalcomposition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

Methods of treating a subject wherein the subject has or is suspected ofhaving breast cancer, lung cancer, prostate cancer, colon cancer, livercancer or melanoma characterized by increased COX-2 protein or nucleicacid, increased COX-2 activity and/or increased Akt activity compared toa control are provided according to aspects of the present inventionwhich include administering a therapeutically effective amount of apharmaceutical composition including a selenium-containing COX-2inhibitor. The subject is a human according to aspects of the presentinvention. Optionally, the pharmaceutical composition is formulated as atopical composition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

According to aspects of the present invention, methods of treating asubject wherein the subject has or is suspected of having breast cancer,lung cancer, prostate cancer, colon cancer, liver cancer or melanomacharacterized by increased COX-2 protein or nucleic acid, increasedCOX-2 activity and/or increased Akt activity compared to a controlinclude administering a therapeutically effective amount of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof. Thesubject is a human according to aspects of the present invention.Optionally, the pharmaceutical composition is formulated as a topicalcomposition. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition.

Administering a therapeutically effective amount of a pharmaceuticalcomposition including a selenium-containing COX-2 inhibitor to a subjecthaving or suspected of having cancer according to aspects of methods oftreatment of the present invention detectably increases apoptosis and/ordecreases proliferation of cells of the cancer and has negligible effecton non-cancer cells. Further, administering a therapeutically effectiveamount of a pharmaceutical composition including a selenium-containingCOX-2 inhibitor to the subject having or suspected of having canceraccording to aspects of methods of treatment of the present inventiondetectably decreases free radicals and/or reactive oxygen species ofcells of the cancer and has negligible effect on non-cancer cells.

Administering a therapeutically effective amount of a pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, to a subject having orsuspected of having cancer according to aspects of methods of treatmentof the present invention detectably increases apoptosis and/or decreasesproliferation of cells of the cancer and has negligible effect onnon-cancer cells. Further, administering a therapeutically effectiveamount of a pharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof, tothe subject having or suspected of having cancer according to aspects ofmethods of treatment of the present invention detectably decreases freeradicals and/or reactive oxygen species of cells of the cancer and hasnegligible effect on non-cancer cells.

Administering a therapeutically effective amount of a pharmaceuticalcomposition including a selenium-containing COX-2 inhibitor to a subjecthaving or suspected of having breast cancer, lung cancer, prostatecancer, colon cancer, liver cancer or melanoma according to aspects ofmethods of treatment of the present invention detectably increasesapoptosis and/or decreases proliferation of cells of the cancer and hasnegligible effect on non-cancer cells. Further, administering atherapeutically effective amount of a pharmaceutical compositionincluding a selenium-containing COX-2 inhibitor to the subject having orsuspected of having breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma according to aspects of methods oftreatment of the present invention detectably decreases free radicalsand/or reactive oxygen species of cells of the cancer and has negligibleeffect on non-cancer cells.

Administering a therapeutically effective amount of a pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, to a subject having orsuspected of having breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma according to aspects of methods oftreatment of the present invention detectably increases apoptosis and/ordecreases proliferation of cells of the cancer and has negligible effecton non-cancer cells. Further, administering a therapeutically effectiveamount of a pharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof tothe subject having or suspected of having breast cancer, lung cancer,prostate cancer, colon cancer, liver cancer or melanoma according toaspects of methods of treatment of the present invention detectablydecreases free radicals and/or reactive oxygen species of cells of thecancer and has negligible effect on non-cancer cells.

Selenium-containing COX-2 inhibitor compounds of the present inventiontarget COX-2 and Akt signaling in cancer cells such that expressionand/or activity of these proteins or any downstream member of thesesignaling cascades is used to identify patients in need of treatmentwith selenium-containing COX-2 inhibitor compounds of the presentinvention, and/or to measure the therapeutic efficacy of such treatment.Thus, according to aspects of the present invention, methods of treatinga subject having or suspected of having cancer such as, but not limitedto, breast cancer, lung cancer, prostate cancer, colon cancer, livercancer and melanoma, includes assaying assaying COX-2 protein, COX-2nucleic acid, COX-2 activity and/or Akt activity in cells of the canceror putative cancer, prior to and/or after administration of aselenium-containing COX-2 inhibitor of the present invention to thesubject.

According to aspects of the present invention, methods of treating asubject having or suspected of having cancer such as, but not limitedto, breast cancer, lung cancer, prostate cancer, colon cancer, livercancer and melanoma, includes assaying assaying COX-2 protein, COX-2nucleic acid, COX-2 activity and/or Akt activity in cells of the canceror putative cancer, prior to and/or after administration ofselenocoxib-1-GSH, selenocoxib-1-cysteine, selenocoxib-1-NAC or two ormore of selenocoxib-1-GSH, selenocoxib-1-cysteine and selenocoxib-1-NAC,to the subject.

Methods of treating a subject having or suspected of having canceraccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity in cellsof the cancer prior to and/or after administration of a pharmaceuticalcomposition including a selenium-containing COX-2 inhibitor to thesubject to determine the level of expression of COX-2 protein in thecells of the cancer compared to a control, the level of expression ofCOX-2 nucleic acid in the cells of the cancer compared to a control, thelevel of activity of COX-2 in the cells of the cancer compared to acontrol and/or the level of activity of Akt in the cells of the cancercompared to a control.

Methods of treating a subject having or suspected of having canceraccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity in cellsof the cancer prior to and/or after administration of a pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, to the subject to determinethe level of expression of COX-2 protein in the cells of the cancercompared to a control, the level of expression of COX-2 nucleic acid inthe cells of the cancer compared to a control, the level of activity ofCOX-2 in the cells of the cancer compared to a control and/or the levelof activity of Akt in the cells of the cancer compared to a control.

Methods of treating a subject having or suspected of having breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma according to aspects of the present invention include assayingCOX-2 protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity incells of the breast cancer, lung cancer, prostate cancer, colon cancer,liver cancer or melanoma prior to and/or after administration of apharmaceutical composition including a selenium-containing COX-2inhibitor to the subject to determine the level of expression of COX-2protein in the cells of the breast cancer, lung cancer, prostate cancer,colon cancer, liver cancer or melanoma compared to a control, the levelof expression of COX-2 nucleic acid in the cells of the breast cancer,lung cancer, prostate cancer, colon cancer, liver cancer or melanomacompared to a control, the level of activity of COX-2 in the cells ofthe breast cancer, lung cancer, prostate cancer, colon cancer, livercancer or melanoma compared to a control and/or the level of activity ofAkt in the cells of the breast cancer, lung cancer, prostate cancer,colon cancer, liver cancer or melanoma compared to a control.

Methods of treating a subject having or suspected of having breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma according to aspects of the present invention include assayingCOX-2 protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity incells of the breast cancer, lung cancer, prostate cancer, colon cancer,liver cancer or melanoma prior to and/or after administration of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof, tothe subject to determine the level of expression of COX-2 protein in thecells of the breast cancer, lung cancer, prostate cancer, colon cancer,liver cancer or melanoma compared to a control, the level of expressionof COX-2 nucleic acid in the cells of the breast cancer, lung cancer,prostate cancer, colon cancer, liver cancer or melanoma compared to acontrol, the level of activity of COX-2 in the cells of the breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma compared to a control and/or the level of activity of Akt inthe cells of the breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma compared to a control.

Methods of treating a subject having or suspected of having canceraccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activity incells of the cancer prior to and/or after administration of apharmaceutical composition including a selenium-containing COX-2inhibitor to the subject to determine the level of expression of COX-2protein in the cells of the cancer compared to a control, the level ofexpression of COX-2 nucleic acid in the cells of the cancer compared toa control, the level of activity of COX-2 in the cells of the cancercompared to a control and/or the level of activity of Akt3 in the cellsof the cancer compared to a control.

Methods of treating a subject having or suspected of having canceraccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activity incells of the cancer prior to and/or after administration of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof, tothe subject to determine the level of expression of COX-2 protein in thecells of the cancer compared to a control, the level of expression ofCOX-2 nucleic acid in the cells of the cancer compared to a control, thelevel of activity of COX-2 in the cells of the cancer compared to acontrol and/or the level of activity of Akt3 in the cells of the cancercompared to a control.

Methods of treating a subject having or suspected of having breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma according to aspects of the present invention include assayingCOX-2 protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activityin cells of the breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma prior to and/or after administration ofa pharmaceutical composition including a selenium-containing COX-2inhibitor to the subject to determine the level of expression of COX-2protein in the cells of the breast cancer, lung cancer, prostate cancer,colon cancer, liver cancer or melanoma compared to a control, the levelof expression of COX-2 nucleic acid in the cells of the breast cancer,lung cancer, prostate cancer, colon cancer, liver cancer or melanomacompared to a control, the level of activity of COX-2 in the cells ofthe breast cancer, lung cancer, prostate cancer, colon cancer, livercancer or melanoma compared to a control and/or the level of activity ofAkt3 in the cells of the breast cancer, lung cancer, prostate cancer,colon cancer, liver cancer or melanoma compared to a control.

Methods of treating a subject having or suspected of having breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma according to aspects of the present invention include assayingCOX-2 protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activityin cells of the breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma prior to and/or after administration ofa pharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof tothe subject to determine the level of expression of COX-2 protein in thecells of the breast cancer, lung cancer, prostate cancer, colon cancer,liver cancer or melanoma compared to a control, the level of expressionof COX-2 nucleic acid in the cells of the breast cancer, lung cancer,prostate cancer, colon cancer, liver cancer or melanoma compared to acontrol, the level of activity of COX-2 in the cells of the breastcancer, lung cancer, prostate cancer, colon cancer, liver cancer ormelanoma compared to a control and/or the level of activity of Akt3 inthe cells of the breast cancer, lung cancer, prostate cancer, coloncancer, liver cancer or melanoma compared to a control.

Optionally, the control is a result of assaying a test sample includingcells of the cancer obtained from the subject prior to administration ofthe pharmaceutical composition.

In a further option, an adjunct anti-cancer treatment is alsoadministered to the subject according to aspects of methods of thepresent invention.

Optionally, the pharmaceutical composition is administered topically.

In a further option, an additional therapeutic agent is administered tothe subject.

Methods of treating melanoma in a subject are provided according toaspects of the present invention which include administering atherapeutically effective amount of a pharmaceutical compositionincluding a selenium-containing COX-2 inhibitor. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a subject are provided according toaspects of the present invention which include administering atherapeutically effective amount of selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof.Optionally, the pharmaceutical composition is formulated for topicalapplication. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition. Another option is administration of anadjunct anti-cancer treatment to the subject. In a still further option,the pharmaceutical composition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention which include administering atherapeutically effective amount of a pharmaceutical compositionincluding a selenium-containing COX-2 inhibitor. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention which include administering atherapeutically effective amount of selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof.Optionally, the pharmaceutical composition is formulated for topicalapplication. In a further option, the pharmaceutical compositionincludes a particulate or nanoparticulate pharmaceutically acceptablecarrier. In another option, the pharmaceutical composition includesnanoliposomes having an average particle size in the range of 1-100 nm.In a further option, an additional therapeutic agent is included in thepharmaceutical composition. Another option is administration of anadjunct anti-cancer treatment to the subject. In a still further option,the pharmaceutical composition is administered topically.

Methods of treating melanoma in a subject are provided according toaspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt activitycompared to a control, which include administering a therapeuticallyeffective amount of a pharmaceutical composition including aselenium-containing COX-2 inhibitor. Optionally, the pharmaceuticalcomposition is formulated for topical application. In a further option,the pharmaceutical composition includes a particulate or nanoparticulatepharmaceutically acceptable carrier. In another option, thepharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a subject are provided according toaspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt activitycompared to a control, which include administering a therapeuticallyeffective amount of selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt activitycompared to a control, which include administering a therapeuticallyeffective amount of a pharmaceutical composition including aselenium-containing COX-2 inhibitor. Optionally, the pharmaceuticalcomposition is formulated for topical application. In a further option,the pharmaceutical composition includes a particulate or nanoparticulatepharmaceutically acceptable carrier. In another option, thepharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt activitycompared to a control, which include administering a therapeuticallyeffective amount of selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a subject are provided according toaspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt3 activitycompared to a control, which include administering a therapeuticallyeffective amount of a pharmaceutical composition including aselenium-containing COX-2 inhibitor. Optionally, the pharmaceuticalcomposition is formulated for topical application. In a further option,the pharmaceutical composition includes a particulate or nanoparticulatepharmaceutically acceptable carrier. In another option, thepharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a subject are provided according toaspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt3 activitycompared to a control, which include administering a therapeuticallyeffective amount of selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt3 activitycompared to a control, which include administering a therapeuticallyeffective amount of a pharmaceutical composition including aselenium-containing COX-2 inhibitor. Optionally, the pharmaceuticalcomposition is formulated for topical application. In a further option,the pharmaceutical composition includes a particulate or nanoparticulatepharmaceutically acceptable carrier. In another option, thepharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

Methods of treating melanoma in a human subject are provided accordingto aspects of the present invention, wherein the subject has or issuspected of having melanoma characterized by increased COX-2 protein ornucleic acid, increased COX-2 activity and/or increased Akt3 activitycompared to a control, which include administering a therapeuticallyeffective amount of selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof. Optionally, thepharmaceutical composition is formulated for topical application. In afurther option, the pharmaceutical composition includes a particulate ornanoparticulate pharmaceutically acceptable carrier. In another option,the pharmaceutical composition includes nanoliposomes having an averageparticle size in the range of 1-100 nm. In a further option, anadditional therapeutic agent is included in the pharmaceuticalcomposition. Another option is administration of an adjunct anti-cancertreatment to the subject. In a still further option, the pharmaceuticalcomposition is administered topically.

According to aspects of the present invention, administering thetherapeutically effective amount of the pharmaceutical composition tothe subject detectably increases apoptosis and/or decreasesproliferation of cells of the melanoma and has negligible effect onnon-cancer cells.

According to aspects of the present invention, administering thetherapeutically effective amount of the pharmaceutical composition tothe subject detectably decreases free radicals and/or reactive oxygenspecies of cells of the melanoma and has negligible effect on non-cancercells.

Methods of treating a subject having or suspected of having melanomaaccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity in cellsof the melanoma, prior to and/or after administration of apharmaceutical composition including a selenium-containing COX-2inhibitor to the subject, to determine the level of expression of COX-2protein in the cells of the melanoma compared to a control, the level ofexpression of COX-2 nucleic acid in the cells of the melanoma comparedto a control, the level of activity of COX-2 in the cells of themelanoma compared to a control and/or the level of activity of Akt inthe cells of the melanoma compared to a control.

Methods of treating a subject having or suspected of having melanomaaccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity in cellsof the melanoma prior to and/or after administration of a pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, to the subject to determinethe level of expression of COX-2 protein in the cells of the melanomacompared to a control, the level of expression of COX-2 nucleic acid inthe cells of the melanoma compared to a control, the level of activityof COX-2 in the cells of the melanoma compared to a control and/or thelevel of activity of Akt in the cells of the melanoma compared to acontrol.

Methods of treating a subject having or suspected of having melanomaaccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activity incells of the melanoma prior to and/or after administration of apharmaceutical composition including a selenium-containing COX-2inhibitor to the subject to determine the level of expression of COX-2protein in the cells of the melanoma compared to a control, the level ofexpression of COX-2 nucleic acid in the cells of the melanoma comparedto a control, the level of activity of COX-2 in the cells of themelanoma compared to a control and/or the level of activity of Akt3 inthe cells of the melanoma compared to a control.

Methods of treating a subject having or suspected of having melanomaaccording to aspects of the present invention include assaying COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt3 activity incells of the melanoma prior to and/or after administration of apharmaceutical composition including selenocoxib-1-GSH,selenocoxib-1-cysteine or selenocoxib-1-NAC; or two or more thereof, tothe subject to determine the level of expression of COX-2 protein in thecells of the melanoma compared to a control, the level of expression ofCOX-2 nucleic acid in the cells of the melanoma compared to a control,the level of activity of COX-2 in the cells of the melanoma compared toa control and/or the level of activity of Akt3 in the cells of themelanoma compared to a control.

According to aspects of the present invention, assay of COX-2 protein,COX-2 nucleic acid, COX-2 activity and/or Akt3 activity in cells of themelanoma is performed after administration of the pharmaceuticalcomposition including a selenium-containing COX-2 inhibitor to determinethe level of expression of COX-2 protein in the cells of the melanomacompared to a control, the level of expression of COX-2 nucleic acid inthe cells of the melanoma compared to a control, the level of activityof COX-2 in the cells of the melanoma compared to a control and/or thelevel of activity of Akt3 in the cells of the melanoma compared to atest sample obtained from the subject prior to administration of thepharmaceutical composition. Optionally, the control is a result ofassaying a test sample including cells of the melanoma obtained from thesubject prior to administration of the pharmaceutical composition.

According to aspects of the present invention, assay of COX-2 protein,COX-2 nucleic acid, COX-2 activity and/or Akt3 activity in cells of themelanoma is performed after administration of the pharmaceuticalcomposition including selenocoxib-1-GSH, selenocoxib-1-cysteine orselenocoxib-1-NAC; or two or more thereof, to determine the level ofexpression of COX-2 protein in the cells of the melanoma compared to acontrol, the level of expression of COX-2 nucleic acid in the cells ofthe melanoma compared to a control, the level of activity of COX-2 inthe cells of the melanoma compared to a control and/or the level ofactivity of Akt3 in the cells of the melanoma compared to a test sampleobtained from the subject prior to administration of the pharmaceuticalcomposition. Optionally, the control is a result of assaying a testsample including cells of the melanoma obtained from the subject priorto administration of the pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing elevated levels of COX-2 expression inmelanoma patient tumors and cell lines;

FIG. 1B is a graph and image of a Western blot showing that COX-2expression is increased in a cell line based melanoma tumor progressionmodel;

FIG. 1C is a graph and image of a Western blot showing that targetingCOX-2 using siRNAs decreased melanoma cell viability;

FIG. 1D is an image of a Western blot showing that SiRNA mediatedinhibition of the MAP kinase pathway decreased COX-2 expression inmelanomas;

FIG. 1E is an image of a Western blot showing that that PLX-4032targeting of ^(V600E)B-Raf decreased COX-2 expression in 1205 Lumelanoma cells;

FIG. 1F is an image of a Western blot showing that that PLX-4032targeting of ^(V600E)B-Raf decreased COX-2 expression in UACC 903melanoma cells;

FIG. 1G is an image of a Western blot showing that U0126 targeting ofMek1/2 decreased COX-2 expression in 1205 Lu and UACC 903 melanomacells;

FIG. 2A shows chemical structures of celecoxib, selenocoxib-2 andselenocoxib-1-GSH;

FIG. 2B is a graph showing that the number of inhibited normal cells issignificantly reduced compared to the number of inhibited cancer cellswhen both are treated with selenocoxib-1-GSH;

FIG. 2C is a graph showing that selenocoxib-1-GSH has COX-2 inhibitoryactivity;

FIG. 3A is a graph showing that selenocoxib-1-GSH but not celecoxibinhibits 1205 Lu and UACC 903 melanoma cell viability;

FIG. 3B is a graph showing that selenocoxib-1-GSH reduces proliferationof 1205 Lu and UACC 903 melanoma cells;

FIG. 3C is a graph showing that selenocoxib-1-GSH increases caspase-3/7activity, which is an indicator of apoptosis, in 1205 Lu and UACC 903melanoma cells;

FIG. 3D is a graph showing that selenocoxib-1-GSH arrests melanoma cellsin the G₀-G₁ phase of the cell cycle;

FIG. 4A is a graph showing that siRNA-mediated targeting of COX-2decreases melanoma tumor development in mice;

FIG. 4B is a graph showing a significant decrease in xenograft tumordevelopment compared to vehicle DMSO treated mice is observed from day16 in 1205 Lu tumors, and a decrease in tumor volume is observed,following selenocoxib-1-GSH treatment compared to controls, with nonoticeable changes in animal body weight;

FIG. 4C is a graph showing a significant decrease in xenograft tumordevelopment compared to vehicle DMSO treated mice is observed in UACC903 tumors from day 22, and a decrease in tumor volume is observed,following selenocoxib-1-GSH treatment compared to controls, with nonoticeable changes in animal body weight;

FIG. 4D is a graph showing that selenocoxib-1-GSH does not affect bloodbiomarkers indicative of major organ related toxicity;

FIG. 5 shows images of H&E stained tissue sections from control DMSOvehicle or selenocoxib-1-GSH treated mice showing no changes in themorphology or architecture of the liver, heart, lung, kidney or spleenand demonstrating that selenocoxib-1-GSH effectively inhibits melanomatumor development leading to tumor regression without significant organrelated toxicity;

FIG. 6A is a graph showing that selenocoxib-1-GSH inhibits the PI3K/Aktsignaling pathway;

FIG. 6B is a graph showing that selenocoxib-1-GSH activates the MAPKsignaling pathway

FIG. 6C is a graph showing that selenocoxib-1-GSH inhibits proteinindicating cell proliferation;

FIG. 6D is a graph showing that selenocoxib-1-GSH increased proteincleavage indicating apoptosis;

FIG. 7A is a graph showing that selenocoxib-1-GSH more dramaticallydecreased ROS compared to celecoxib and selenocoxib-1 in UACC 903melanoma cells; and

FIG. 7B is a graph showing that selenocoxib-1-GSH more dramaticallydecreased ROS compared to celecoxib and selenocoxib-1 in 1205 Lumelanoma cells.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods are provided by the present invention toinhibit COX-2 activity in cancer cells, reduce survival and/or reduceproliferation of cancer cells.

The singular terms “a,” “an,” and “the” used herein include pluralreferents unless the context clearly indicates otherwise.

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Chu, E. and Devita, V. T., Eds., Physicians' Cancer ChemotherapyDrug Manual, Jones & Bartlett Publishers, 2005; J. M. Kirkwood et al.,Eds., Current Cancer Therapeutics, 4th Ed., Current Medicine Group,2001; Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins, 21st Ed., 2006; L. V. Allen, Jr. et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed.,Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; and L. Bruntonet al., Goodman & Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill Professional, 11th Ed., 2005.

Anti-cancer compositions and methods are provided according to aspectsof the present invention. According to aspects of the present invention,the present invention relates to inventive selenium-containing COX-2inhibitors, methods for treatment and/or prevention of cancer in asubject using one or more selenium-containing COX-2 inhibitors.

According to aspects of the present invention, a selenium-containingCOX-2 inhibitor compound included in methods and compositions of theinvention is selenocoxib-1-glutathione (selenocoxib-1-GSH),selenocoxib-1-cysteine and/or selenocoxib-1-N-aceylcysteine(selenocoxib-1-NAC).

Selenocoxib-1-GSH has the structural formula:

Selenocoxib-1-cysteine has the structural formula:

Selenocoxib-1-N-aceylcysteine has the structural formula:

Compositions and methods according to aspects of the present inventionprevent and inhibit cancer cell proliferation and tumor development andare considered useful as chemotherapeutic and chemopreventive agents.

Methods and compositions are provided according to the present inventionfor treating cancer. Particular cancers treated using methods andcompositions described herein are characterized by abnormal cellproliferation including, but not limited to, pre-neoplastichyperproliferation, cancer in-situ, neoplasms, metastases, tumors,benign growths or other abnormal cell proliferation condition responsiveto a composition of the present invention. Methods and compositions ofthe present invention can be used for prophylaxis as well asamelioration of signs and/or symptoms of cancer. The terms “treating”and “treatment” used to refer to treatment of a cancer in a subjectinclude: preventing, inhibiting or ameliorating the cancer in thesubject, such as slowing progression of the cancer and/or reducing orameliorating a sign or symptom of the cancer.

A therapeutically effective amount of a composition of the presentinvention is an amount which has a beneficial effect in a subject beingtreated. In subjects having cancer or at risk for having cancer, such asa condition characterized by abnormal cell proliferation including, butnot limited to, pre-neoplastic hyperproliferation, cancer in-situ,neoplasms, metastasis, a tumor, a benign growth or other conditionresponsive to a composition of the present invention, a therapeuticallyeffective amount of a composition of the present invention is effectiveto ameliorate or prevent one or more signs and/or symptoms of thecondition. For example, a therapeutically effective amount of acomposition of the present invention is effective to detectably increaseapoptosis and/or decrease proliferation of cells of a cancer conditioncharacterized by abnormal cell proliferation including, but not limitedto, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, a tumor, a benign growth or other abnormal cellproliferation condition responsive to a composition of the presentinvention, without significant effect to increase apoptosis or decreaseproliferation of non-cancer cells.

In particular aspects, cancers treated using methods and compositions ofthe present invention are characterized by elevated expression and/oractivity of COX-2. Elevated expression and/or activity of COX-2 has beenreported in cancers of the prostate, breast, colon, kidney, liver andmelanocyte, Ghosh N et al., Pharmacol Rep, 2010; 62:233-44; Becker M Ret al., Melanoma Res, 2009; 19:8-16; and Flockhart R J et al., Br JCancer, 2009; 101:1448-55. Treatment of a cancer characterized byelevated expression and/or activity of COX-2 using methods andcompositions of the present invention results in a detectable decreasein expression and/or activity of COX-2. Assays for expression and/oractivity of COX-2 which identify cancers amenable to treatment accordingto the present invention and which allow for monitoring of effectivenessof treatment include, but are not limited to immunoassays, nucleic acidassays and enzyme activity assays.

The term “expression” refers to transcription of a gene to produce acorresponding mRNA and/or translation of the mRNA to produce thecorresponding protein.

In particular aspects, cancers treated using methods and compositions ofthe present invention are characterized by Akt dysregulation.

Akt, a serine/threonine protein kinase also known as protein kinase B,has a stimulatory effect on cell cycle progression, cell proliferationand inhibition of apoptosis. Akt proteins, nucleic acids and signalingpathway components are described, for instance, see. Testa, J. R. etal., PNAS, 98:10983-10985; Fayard, E. et al., J. Cell Sci.,118:5675-5678, 2005; Cheng, J. and S. Nicosia, (2001) AKT signaltransduction pathway in oncogenesis, in Encyclopedic Reference ofCancer, D. Schwab, Editor. 2001, Springer: Berlin, Germany, p. 35-7;Datta, S. R., et al. (1999) Cellular survival: a play in three Akts.Genes Dev, 13(22): 2905-27; Fayard, E. et al. (2005) J Cell Sci, 118(Pt24: 5675-8; Mirza, A. M., Fayard, E. et al. (2000) 2000. 11(6: 279-92;Nicholson, K. M. and N. G. Anderson, (2002) Cell Signal, 2002, 14(5): p.381-95; Paez, J. and W. Sellers, (2003) PI3K/PTEN/Akt Pathway: ACritical Mediator of Oncogenic Signaling, in Signal Transduction inCancer, D. Frank, Editor. 2003, Kluwer Academic Publishers: Netherlands;and Testa, J. R.; P. N. Tsichlis, (2005) Oncogene, 24(50): 7391-3 andother references listed herein.

Akt family members, Akt1, Akt2 and Akt3, are activated byphosphorylation, membrane translocation, increases in gene copy numberand/or loss of a negative regulatory phosphatase, PTEN. Increasedactivation of Akt, including increased levels of Akt and/or increasedlevels of phosphorylated Akt is an indicator of Akt dysregulationassociated with proliferation and cell survival in pathogenicconditions, such as cancer.

Akt3 is active in ˜70% of melanomas. While all three Akt isoforms areexpressed in melanocytes and melanoma cells, Akt3 is the predominantlyactive family member. Dysregulated Akt3 activity in melanoma cellsreduces cellular apoptosis mediated through caspase-3, thereby promotingmelanoma tumor development.

Treatment of a cancer characterized by dysregulation of Akt usingmethods and compositions of the present invention results in adetectable decrease in expression and/or activity of Akt. In particular,treatment of a cancer characterized by dysregulation of Akt3 usingmethods and compositions of the present invention results in adetectable decrease in expression and/or activity of Akt3. Aktdysregulation is determined, for instance, by measurement of Akt genecopy number, Akt protein or RNA levels and/or levels of phosphorylatedAkt, in cells known or suspected to be dysplasic, pre-cancerous,cancerous, metastatic or otherwise characterized by abnormal cellproliferation compared to normal cells. Assays for Akt dysregulationwhich identify cancers amenable to treatment according to the presentinvention and which allow for monitoring of effectiveness of treatmentinclude, but are not limited to immunoassays and nucleic acid assays.

Methods of treating a subject are provided according to aspects of thepresent invention which include administering a therapeuticallyeffective amount of an inventive selenium-containing COX-2 inhibitorcompound wherein the subject has a condition characterized by Aktdysregulation and/or elevated expression and/or activity of COX-2, suchas cancer, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, tumor or benign growth. In certain aspects of methods oftreatment of a subject, contacting cells characterized by Aktdysregulation with a therapeutic amount of an inventiveselenium-containing COX-2 inhibitor compound decreases a component of anAkt signaling pathway selected from the group consisting of: an Akt1signaling pathway; an Akt2 signaling pathway; an Akt3 signaling pathway;and a combination thereof. For example, contacting the cell with aninventive selenium-containing COX-2 inhibitor compound decreases acomponent of an Akt signaling pathway selected from pAkt1, pAkt2, pAk3,pPRAS40 and a combination thereof.

Since selecoxib-1-GSH, and other inventive selenium-containing COX-2inhibitor compounds of the present invention, targets COX-2 and Aktsignaling in melanoma cells and other cancer types, expression oractivity of these proteins or any downstream member of these signalingcascades could be used to identify patients that could be treated withselecoxib-1-GSH, and other inventive selenium-containing COX-2 inhibitorcompounds of the present invention, or to measure the therapeuticefficacy thereof. For example, in melanoma the expression or activity ofAkt3 or downstream PRAS40 would be measured, prior to and/or followingtreatment with selecoxib-1-GSH, and other inventive selenium-containingCOX-2 inhibitor compounds of the present invention. Other downstreammembers of these pathways could be assessed in a similar fashion.

In addition to direct measurement of protein levels and/or activity ofCOX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3, one or moredownstream components of the signaling pathways of these can bemeasured.

In aspects of described methods, treatment of a subject with atherapeutically effective amount an inventive selenium-containing COX-2inhibitor compound is substantially without toxic effect on cells inwhich Akt is not dysregulated and cells without elevated expressionand/or activity of COX-2.

Methods of treating a subject are provided according to aspects of thepresent invention which include administering a therapeuticallyeffective amount of a selenium-containing COX-2 inhibitor and whereinthe subject has a condition characterized by Akt dysregulation and/orelevated expression and/or activity of COX-2, such as cancer,pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, tumor or benign growth.

In particular aspects, cancers treated using methods and compositions ofthe present invention are characterized by Akt dysregulation, elevatedCOX-2 expression and/or elevated activity of COX-2, including, but notlimited to, breast cancer, lung cancer, prostate cancer, colon cancer,liver cancer and melanoma. Akt dysregulation, elevated COX-2 expressionand/or elevated activity of COX-2 can be detected by assaying a sample,such as a biopsy sample, obtained from a subject having or suspected ofhaving cancer.

Aspects of the present invention include assaying one or more of COX-2,Akt1, Akt2, Akt3, pAkt1 (phosphorylated Akt1), pAkt2 (phosphorylatedAkt2) and pAkt3 (phosphorylated Akt3) protein in a test sample obtainedfrom a subject having or suspected of having cancer.

In addition to direct measurement of expression and/or activity ofCOX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3, one or moredownstream components of the signaling pathways of these can bemeasured.

Phosphorylation of the downstream Akt3 substrate PRAS40 is significantlyinhibited by treatment of a cancer with a selenium-containing COX-2inhibitor of the present invention. For example, phosphorylation of thedownstream Akt3 substrate PRAS40 is significantly inhibited by treatmentof a cancer with selenocoxib-1-GSH of the present invention.

Aspects of the present invention include assaying one or more of COX-2,Akt1, Akt2, Akt3, pAkt1, pAkt2 and pAkt3 protein in a test sampleobtained from a subject having or suspected of having melanoma.

Aspects of the present invention include assaying one or more of COX-2,Akt3, and pAkt3 proteins in a test sample obtained from a subject havingor suspected of having melanoma.

Aspects of the present invention include assaying one or more of COX-2,Akt1, Akt2 and Akt3 nucleic acids in a test sample obtained from asubject having or suspected of having cancer.

Aspects of the present invention include assaying one or more of COX-2,Akt1, Akt2 and Akt3 nucleic acids in a test sample obtained from asubject having or suspected of having melanoma.

A test sample can be any biological fluid, cell or tissue of a subjectcontaining or suspected of containing cancer cells, illustrativelyincluding blood, plasma, urine, saliva, ascites, cerebrospinal fluid,cerebroventricular fluid, pleural fluids, pulmonary and bronchial lavagesamples, mucous, sweat, tears, semen, bladder wash samples, amnioticfluid, lymph, peritoneal fluid, synovial fluid, bone marrow aspirate,tumor cells or tissue, organ cells or tissue, such as biopsy material.

Immunoassay methods can be used to assay any one or more proteins suchas COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and pAkt3 in a sample.Immunoassays that can be used include but are not limited to,enzyme-linked immunosorbent assay (ELISA), enzyme-linkedimmunofiltration assay (ELIFA), flow cytometry, immunoblot,immunoprecipitation, immunohistochemistry, immunocytochemistry,luminescent immunoassay (LIA), fluorescent immunoassay (FIA), andradioimmunoassay. Assay methods may be used to obtain qualitative and/orquantitative results. A method to assay one or more proteins such asCOX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and pAkt3 is to isolatecirculating tumor cells and probe the fixed cells with quantum dots orsimilar imaging moieties conjugated to antibodies thereby measuringlevels of COX-2 protein or the presence of pAKT in the cells, allowingfor simultaneous measurement of the levels of two or more proteins inthe same cell. Comparison to normal control cells is performed to assesstumor status and/or drug efficacy.

Specific details of suitable assay methods for both qualitative andquantitative assay of a sample are described in standard references,illustratively including E. Harlow and D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling and S.Dübel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H.Zola, Monoclonal Antibodies: Preparation and Use of MonoclonalAntibodies and Engineered Antibody Derivatives, Basics: From Backgroundto Bench, BIOS Scientific Publishers, 2000; B. K. C. Lo, AntibodyEngineering Methods and Protocols, Methods in Molecular Biology, HumanaPress, 2003; F. M. Ausubel et al., Eds., Short Protocols in MolecularBiology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The AptamerHandbook: Functional Oligonucleotides and Their Applications, Wiley,2006; Ormerod, M. G., Flow Cytometry: a practical approach, OxfordUniversity Press, 2000; Givan, A. L., Flow Cytometry: First Principles,Wiley, New York, 2001; Gorczyca, W., Flow Cytometry in NeoplasticHematology: morphologic-immunophenotypic correlation, Taylor & Francis,2006; Crowther, J. R., The ELISA Guidebook (Methods in MolecularBiology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, 3rdEdition, Elsevier Science, 2005 and J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 3rd Ed., 2001.

Antibodies directed against COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 orpAkt3 can be polyclonal or monoclonal antibodies. Suitable antibodiesalso include chimeric antibodies, humanized antibodies, and proteinbinding antibody fragments and molecules having specific protein bindingfunctionality, such as aptamers. Examples of antibody fragments that canbe used in embodiments of inventive assays include Fab fragments, Fab′fragments, F(ab′)₂ fragments, Fd fragments, Fv fragments, scFvfragments, and domain antibodies (dAb).

Antibodies and methods for preparation of antibodies are well-known inthe art. Details of methods of antibody generation and screening ofgenerated antibodies for substantially specific binding to an antigenare described in standard references such as E. Harlow and D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1988; F. Breitling and S. Dübel, Recombinant Antibodies, John Wiley &Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation andUse of Monoclonal Antibodies and Engineered Antibody Derivatives,Basics: From Background to Bench, BIOS Scientific Publishers, 2000; andB. K. C. Lo, Antibody Engineering: Methods and Protocols, Methods inMolecular Biology, Humana Press, 2003.

Aptamers can be used to assay a sample for proteins such as COX-2, Akt1,Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3. The term “aptamer” refers to apeptide and/or nucleic acid that substantially specifically binds to aspecified substance. In the case of a nucleic acid aptamer, the aptameris characterized by binding interaction with a target other thanWatson/Crick base pairing or triple helix binding with a second and/orthird nucleic acid. Such binding interaction may include Van der Waalsinteraction, hydrophobic interaction, hydrogen bonding and/orelectrostatic interactions, for example. Similarly, peptide-basedaptamers are characterized by specific binding to a target wherein theaptamer is not a naturally occurring ligand for the target. Techniquesfor identification and generation of peptide and nucleic acid aptamersand their use are known in the art as described, for example, in F. M.Ausubel et al., Eds., Short Protocols in Molecular Biology, CurrentProtocols, Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook:Functional Oligonucleotides and Their Applications, Wiley, 2006; and J.Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 3rd Ed., 2001.

Optionally, spectrometric analysis is used to assay a sample forproteins such as COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3. Forexample mass analysis can be used in an assay according to embodimentsof the present invention. Mass analysis is conducted using, for example,time-of-flight (TOP) mass spectrometry or Fourier transform ioncyclotron resonance mass spectrometry. Mass spectrometry techniques areknown in the art and exemplary detailed descriptions of methods forprotein and/or peptide assay are found in Li J., et al., Clin Chem.,48(8):1296-304, 2002; Hortin, G. L., Clinical Chemistry 52: 1223-1237,2006; Hortin, G. L., Clinical Chemistry 52: 1223-1237, 2006; A. L.Burlingame, et al. (Eds.), Mass Spectrometry in Biology and Medicine,Humana Press, 2000; and D. M. Desiderio, Mass Spectrometry of Peptides,CRC Press, 1990.

Assay of proteins such as COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/orpAkt3 can be performed on cells and tissues.

One or more standards can be used to allow quantitative determination ofproteins such as COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3 in asample.

Assay of proteins such as COX-2, Akt1, Akt2, Akt3, pAkt1, pAkt2 and/orpAkt3 in a test sample is optionally compared to assay of the sameprotein in a control sample. Control samples may be obtained from one ormore normal subjects, for example.

According to embodiments of the present invention, assays for COX-2,Akt1, Akt2, Akt3, pAkt1, pAkt2 and/or pAkt3 are used to monitor diseaseprogression and/or effectiveness of treatment of a subject. Thus, forexample, a test sample is obtained from the subject before treatment ofcancer and at one or more times during and/or following treatment inorder to assess effectiveness of the treatment. In a further example, atest sample is obtained from the subject at various times in order toassess the course or progress of disease or healing.

An assay according to embodiments of the present invention detectsCOX-2, Akt1, Akt2, and/or Akt3 nucleic acid in a test sample of asubject known to have cancer, suspected of having a particular diseaseor injury or at risk of having a particular disease or injury.

Assays for detecting COX-2, Akt1, Akt2, and/or Akt3 nucleic acid,particularly mRNA or cDNA, include, but are not limited to, polymerasechain reactions (PCR) such as RT-PCR, dot blot, in situ hybridization,Northern blot and RNase protection.

COX-2 activity can be determined using a COX-2 enzyme activity assay.COX-2 activity can be measured using any of various assays such as acommercially available fluorometric assay for COX-1 (cyclooxygenase-1)and COX-2 isozymes using the peroxide-sensitive dye AMPLEX Red reagent,10-acetyl-3,7-dihydroxyphenoxazine. Activity of both COX-1 and COX-2 tooxidize 10-acetyl-3,7-dihydroxyphenoxazine in the presence ofarachidonic acid, producing the highly fluorescent dye resorufin allowsfor detection of both COX-1 and COX-2 isozymes and use of selectiveinhibitors of COX-1 or COX-2 allows determination of the activity ofeach enzyme individually. Selective inhibitors of COX-2 are well knownand include, for example, celecoxib. Selective inhibitors of COX-1 arewell known and include, for example aspirin, FR122047, mofezolac, P6,SC-560 and TFAP.

A test sample from a subject is optionally purified for assay accordingto a method of the present invention. The term “purified” in the contextof a test sample refers to separation of COX-2, Akt1, Akt2, Akt3, pAkt1,pAkt2 and/or pAkt3 and/or a nucleic acid encoding COX-2, Akt1, Akt2,and/or Akt3 from at least one other component present in the testsample. Test sample purification is achieved by techniquesillustratively including electrophoretic methods such as gelelectrophoresis and 2-D gel electrophoresis; chromatography methods suchas HPLC, ion exchange chromatography, affinity chromatography, sizeexclusion chromatography, thin layer and paper chromatography. Detailsof these and other techniques for nucleic acid assay are known in theart, for example, as described in J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 3rd Ed., 2001; and F. M. Ausubel, Ed., Short Protocols inMolecular Biology, Current Protocols; 5th Ed., 2002.

A method of treating a subject is provided according to aspects of thepresent invention which includes administering to a subject in needthereof a therapeutically effective amount of one or moreselenium-containing COX-2 inhibitors.

According to aspects of the invention, a method of treating a subjectincludes administering an effective amount of selenocoxib-1-GSH.

According to aspects of the invention, a method of treating a subjectincludes administering an effective amount of selenocoxib-1-cysteine.

According to aspects of the invention, a method of treating a subject,includes administering an effective amount ofselenocoxib-1-N-aceylcysteine.

According to aspects, two or more selenium-containing COX-2 inhibitorsare administered to a subject in need thereof. The two or moreselenium-containing COX-2 inhibitors can be administered (1) as apharmaceutical composition that includes the two or moreselenium-containing COX-2 inhibitors of the present invention together;and/or (2) by co-administration of the two or more selenium-containingCOX-2 inhibitors of the present invention where the selenium-containingCOX-2 inhibitors have not been formulated in the same composition. Whenusing separate formulations, the two or more selenium-containing COX-2inhibitors of the present invention may be administered at the sametime, intermittent times, staggered times, prior to, subsequent to, orcombinations thereof.

According to aspects of the invention, an administeredselenium-containing COX-2 inhibitor is a pharmaceutically acceptablesalt, ester, amide, solvate or stereoisomer of a selenium-containingCOX-2 inhibitor.

According to aspects of the invention, an administeredselenium-containing COX-2 inhibitor is a pharmaceutically acceptablesalt, ester, amide, solvate or stereoisomer of selenocoxib-1-GSH.

According to aspects of the invention, an administeredselenium-containing COX-2 inhibitor is a pharmaceutically acceptablesalt, ester, amide, solvate or stereoisomer of selenocoxib-1-cysteine orselenocoxib-1-N-acetylcysteine.

A “pharmaceutically acceptable” salt, ester, amide or solvate issuitable for use in a subject without undue toxicity or irritation tothe subject and is effective for their intended use.

Pharmaceutically acceptable salts include pharmaceutically acceptableacid addition salts and base addition salts. Pharmaceutically acceptablesalts are well-known in the art, such as those detailed in S. M. Bergeet al., J. Pharm. Sci., 66:1-19, 1977. Exemplary pharmaceuticallyacceptable salts are those suitable for use in a subject without unduetoxicity or irritation to the subject and which are effective for theirintended use which are formed with inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid,sulfuric acid and sulfamic acid; organic acids such as acetic acid,adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonicacid, benzoic acid, 2-acetoxybenzoic acid, butyric acid, camphoric acid,camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid,ethanesulfonic acid, formic acid, fumaric acid, glutamic acid, glycolicacid, glycerophosphoric acid, hemisulfic acid, heptanoic acid, hexanoicacid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid,maleic acid, hydroxymaleic acid, malic acid, malonic acid, mandelicacid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonicacid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoicacid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, picricacid, pivalic acid, propionic acid, pyruvic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaricacid, p-toluenesulfonic acid, trichloroacetic acid, trifluoroacetic acidand undecanoic acid; inorganic bases such as ammonia, hydroxide,carbonate, and bicarbonate of ammonium; organic bases such as primary,secondary, tertiary and quaternary amine compounds ammonium, arginine,betaine, choline, caffeine, diolamine, diethylamine, diethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, N,N′-dibenzylethylenediamine, ethanolamine, ethylamine,ethylenediamine, glucosamine, histidine, hydrabamine, isopropylamine,1H-imidazole, lysine, methylamine, N-ethylpiperidine,N-methylpiperidine, N-methylmorpholine, N,N-dimethylaniline, piperazine,trolamine, methylglucamine, purines, piperidine, pyridine, theobromine,tetramethylammonium compounds, tetraethylammonium compounds,trimethylamine, triethylamine, tripropylamine and tributylamine andmetal cations such as aluminum, calcium, copper, iron, lithium,magnesium, manganese, potassium, sodium, and zinc.

Pharmaceutically acceptable solvates illustratively include hydrates,ethanolates, methanolates.

Exemplary pharmaceutically acceptable amides include amides derived fromammonia, primary C1-C6 alkyl amines and secondary C1-C6 dialkyl aminesincluding those in the form of a 5- or 6-member nitrogen-containingheterocycle.

Compositions including a selenium-containing COX-2 inhibitor areprovided according to aspects of the present invention which inhibittumor growth by inhibiting an Akt signaling cascade and/or inhibitingCOX-2 in cells characterized by Akt dysregulation and/or elevated COX-2expression and/or elevated COX-2 activity.

Methods including administration of one or more selenium-containingCOX-2 inhibitors to a subject in need thereof are provided according toparticular aspects of the present invention which have utility, forexample, in inhibiting the Akt signaling cascade and/or COX-2 in cancercells.

Methods including administration of one or more selenium-containingCOX-2 inhibitors to a subject in need thereof are provided according toparticular aspects of the present invention which have utility, forexample, in decreasing levels of free radicals and/or reactive oxygenspecies (ROS) in cancer cells. ROS can be measured by methods includingbut not limited to using an indicator such as 2′, 7′-dichlorfluoresceinor dichlorodihydrofluorescein diacetate as described herein.

Inhibitors of the Akt signaling cascade and/or COX-2 according toaspects of the present invention have utility in treatment of subjecthaving cancer or at risk of having cancer in which Akt deregulationand/or elevated expression and/or activity of COX-2 occurs, such as inmelanoma and other cancers including, but not limited to, cancers of theprostate, breast, colon, kidney, lung and liver.

Methods of modulating an Akt protein, such as an Akt1, Akt2 and/or anAkt3 protein, in a cell are provided according to aspects of the presentinvention which include contacting the cell with an effective amount ofa selenium-containing COX-2 inhibitor.

Pharmaceutical compositions including a selenium-containing COX-2inhibitor of the present invention are provided according to aspects ofthe present invention.

A pharmaceutical composition includes a selenium-containing COX-2inhibitor of the present invention and a pharmaceutically acceptablecarrier according to aspects of the present invention. The term“pharmaceutically acceptable carrier” refers to a carrier which issubstantially non-toxic to a subject to which the composition isadministered and which is substantially chemically inert with respect toa selenium-containing COX-2 inhibitor of the present invention.

A pharmaceutical composition according to the invention generallyincludes about 0.1-99% of a selenium-containing COX-2 inhibitor.Combinations of selenium-containing COX-2 inhibitors in a pharmaceuticalcomposition are also considered within the scope of the presentinvention.

Combination Treatments

Combinations of therapeutic agents are administered according to aspectsof the present invention. In some aspects, two or moreselenium-containing COX-2 inhibitors of the present invention areadministered to a subject to treat cancer in a subject in need thereof.In further aspects, at least one selenium-containing COX-2 inhibitor ofthe present invention and at least one additional therapeutic agent areadministered to a subject to treat cancer in a subject in need thereof.In still further aspects, at least one selenium-containing COX-2inhibitor of the present invention and at least two additionaltherapeutic agents are administered to a subject to treat cancer in asubject in need thereof.

The term “additional therapeutic agent” is used herein to denote achemical compound, a mixture of chemical compounds, a biologicalmacromolecule (such as a nucleic acid, an antibody, a protein or portionthereof, e.g., a peptide), or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues which is a biologically, physiologically, orpharmacologically active substance (or substances) that acts locally orsystemically in a subject.

Additional therapeutic agents included in aspects of methods andcompositions of the present invention include, but are not limited to,antibiotics, antivirals, antineoplastic agents, analgesics,antipyretics, antidepressants, antipsychotics, anti-cancer agents,antihistamines, anti-osteoporosis agents, anti-osteonecrosis agents,antiinflammatory agents, anxiolytics, chemotherapeutic agents,diuretics, growth factors, hormones, non-steroidal anti-inflammatoryagents, steroids and vasoactive agents.

Combination therapies utilizing one or more selenium-containing COX-2inhibitors of the present invention and one or more additionaltherapeutic agents may show synergistic effects, e.g., a greatertherapeutic effect than would be observed using either the one or moreselenium-containing COX-2 inhibitors of the present invention or one ormore additional therapeutic agents alone as a monotherapy.

According to aspects, combination therapies include: (1) pharmaceuticalcompositions that include one or more selenium-containing COX-2inhibitors of the present invention in combination with one or moreadditional therapeutic agents; and (2) co-administration of one or moreselenium-containing COX-2 inhibitors of the present invention with oneor more additional therapeutic agents wherein the one or moreselenium-containing COX-2 inhibitors of the present invention and theone or more additional therapeutic agents have not been formulated inthe same composition. When using separate formulations, the one or moreselenium-containing COX-2 inhibitors of the present invention may beadministered at the same time, intermittent times, staggered times,prior to, subsequent to, or combinations thereof, with reference to theadministration of the one or more additional therapeutic agents.

Combination treatments can allow for reduced effective dosage andincreased therapeutic index of the one or more selenium-containing COX-2inhibitors of the present invention and the one or more additionaltherapeutic agents used in methods of the present invention. Synergisticeffects of combination treatments including a selenium-containing COX-2inhibitor of the present invention may be obtained.

In particular aspects of the present invention, synergistic anti-cancereffects of combination treatments including administration ofselenocoxib-1-GSH and administration of plumbagin, also known as5-hydroxy-2-methyl-1,4-naphthoquinone, may be obtained.

In particular aspects of the present invention, synergistic anti-cancereffects of a combination treatment of a subject having or suspected ofhaving melanoma including administration of selenocoxib-1-GSH andadministration of plumbagin may be obtained.

In particular aspects of the present invention, synergistic anti-cancereffects of a combination treatment of a subject having or suspected ofhaving a cancer characterized by increased COX-2 protein or nucleicacid, increased COX-2 activity and/or increased Akt activity includingadministration of selenocoxib-1-GSH and administration of plumbagin maybe obtained.

In particular aspects of the present invention, synergistic anti-cancereffects of a combination treatment of a subject having or suspected ofhaving melanoma including administration of selenocoxib-1-GSH andadministration of plumbagin may be obtained.

In particular aspects synergistic effects of combination treatmentsincluding administration of a nanoparticulate liposomal formulation ofselenocoxib-1-GSH and administration of a nanoparticulate liposomalformulation of an additional anti-cancer agent may be obtained.

According to particular aspects of the present invention, synergisticeffects of combination treatments including administration of ananoparticulate liposomal formulation of selenocoxib-1-GSH andadministration of a nanoparticulate liposomal formulation of plumbaginmay be obtained.

Optionally, a method of treating a subject having cancer or at risk ofhaving cancer further includes an adjunct anti-cancer treatment. Anadjunct anti-cancer treatment can be administration of an anti-canceragent.

Anti-cancer agents are described, for example, in Goodman et al.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed.,Macmillan Publishing Co., 1990.

Anti-cancer agents illustratively include5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), acivicin,aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin,allopurinol, altretamine, ambomycin, ametantrone, amifostine,aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenictrioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin,batimastat, benzodepa, bicalutamide, bisantrene, bisnafide dimesylate,bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin,calusterone, capecitabine, caracemide, carbetimer, carboplatin,carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil,cirolemycin, cisplatin, cladribine, crisnatol mesylate,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate,diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone,duazomycin, edatrexate, eflornithine, elsamitrucin, enloplatin,enpromate, epipropidine, epirubicin, erbulozole, esorubicin,estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine,fenretinide, floxuridine, fludarabine, fluorouracil, fluorocitabine,fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea,idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, includingrecombinant interleukin. II or rIL2), interferon alfa-2a, interferonalfa-2b, interferon alfa-n1, interferon alfa-n3, interferon beta-Ia,interferon gamma-Ib, iproplatin, irinotecan, lanreotide, letrozole,leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol,maytansine, mechlorethamine hydrochlride, megestrol, melengestrolacetate, melphalan, menogaril, mercaptopurine, methotrexate, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel,pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide,pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,plomestane, porfimer, porfiromycin, prednimustine, procarbazine,puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine,simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine,spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide,teroxirone, testolactone, thiamiprine, thioguanine, thiotepa,tiazofurin, tirapazamine, topotecan, toremifene, trestolone,triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard,uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate,vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine,vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate,and zorubicin.

An adjunct anti-cancer treatment can be a radiation treatment of asubject or an affected area of a subject's body.

Pharmaceutical compositions suitable for delivery to a subject may beprepared in various forms illustratively including physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers include water, ethanol, polyols such aspropylene glycol, polyethylene glycol, glycerol, and the like, suitablemixtures thereof; vegetable oils such as olive oil; and injectableorganic esters such as ethyloleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants, such as sodium lauryl sulfate. Additionalcomponents illustratively including a buffer, a solvent, or a diluentmay be included.

Such formulations are administered by a suitable route includingparenteral and oral administration. Administration may include systemicor local injection, and particularly intravenous injection.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and substances similar in nature.Prolonged delivery of an injectable pharmaceutical form can be broughtabout by the use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, one or moreselenium-containing COX-2 inhibitors described herein is admixed with atleast one inert customary excipient (or carrier) such as sodium citrateor dicalcium phosphate or (a) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders, as for example, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as forexample, glycerol, (d) disintegrating agents, as for example, agar-agar,calcium carbonate, plant starches such as potato or tapioca starch,alginic acid, certain complex silicates, and sodium carbonate, (e)solution retarders, as for example, paraffin, (f) absorptionaccelerators, as for example, quaternary ammonium compounds, (g) wettingagents, as for example, cetyl alcohol, glycerol monostearate, andglycols (h) adsorbents, as for example, kaolin and bentonite, and (i)lubricants, as for example, talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.In the case of capsules, tablets, and pills, the dosage forms may alsoinclude a buffering agent.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include a pharmaceuticallyacceptable carrier formulated as an emulsion, solution, suspension,syrup, or elixir. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to a selenium-containing COX-2 inhibitor, maycontain suspending agents, as for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitol esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar or tragacanth,or mixtures of these substances, and the like.

In particular aspects, compositions of the present invention areformulated for topical application. In further particular aspects,compositions of the present invention are formulated for topicalapplication and are characterized by less than 10% absorption of anactive ingredient in the composition into the system of an individualtreated topically. In still further particular aspects, compositions ofthe present invention are formulated for topical application and arecharacterized by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% absorptionof an active ingredient in the composition into the system of anindividual treated topically. Absorption into the system of anindividual can be measured by any of various methods, particularly assayfor the active ingredient, a metabolite and/or a breakdown product ofthe active ingredient in a sample obtained from an individual treatedwith the topical formulation. For example, a blood, plasma or serumsample can be assayed for presence of the active ingredient, ametabolite of the active ingredient and/or a breakdown product of theactive ingredient.

A topical formulation can be an ointment, lotion, cream or gel inparticular aspects. Topical dosage forms such as ointment, lotion, creamor gel bases are described in Remington: The Science and Practice ofPharmacy, 21^(st) Ed., Lippincott Williams & Wilkins, 2006, p. 880-882and p. 886-888; and in Allen, L. V. et al., Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems, 8^(th) Ed., Lippincott Williams& Wilkins, 2005, p. 277-297.

Pharmaceutically acceptable carriers and formulation of pharmaceuticalcompositions are known in the art, illustratively including, but notlimited to, as described in Remington: The Science and Practice ofPharmacy, 21^(st) Ed., Lippincott, Williams & Wilkins, Philadelphia,Pa., 2006; and Allen, L. V. et al., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems, 8^(th) Ed., Lippincott, Williams & Wilkins,Philadelphia, Pa., 2005.

The term subject refers to an individual in need of treatment for apathological condition, particularly cancer, and generally includesmammals and birds, such as, but not limited to, humans, other primates,cats, dogs, cows, horses, rodents, pigs, sheep, goats and poultry.

A pharmaceutical composition according to the present invention issuitable for administration to a subject by a variety of systemic and/orlocal routes including, but not limited to, intravenous, intramuscular,subcutaneous, intraperitoneal, oral, otic, rectal, vaginal, topical,parenteral, pulmonary, ocular, nasal, intratumoral and mucosal.

An inventive composition may be administered acutely or chronically. Forexample, a composition as described herein may be administered as aunitary dose or in multiple doses over a relatively limited period oftime, such as seconds-hours. In a further aspect, administration mayinclude multiple doses administered over a period of days-years, such asfor chronic treatment of cancer.

With regard to administration of a particular inventive composition to amammalian subject, particular exemplary effective dosage ranges withoutsignificant systemic toxicity are described in terms of amounts ofselenium administered via administration of the inventive composition.Thus, for example, when delivered by a parenteral route, such asintraperitoneal or intravenous, an exemplary therapeutically effectivedosage of an inventive composition is in the range of about 1-4 ppmselenium, administered three times per week. It is noted that the doserange “about 1-4 ppm selenium” refers to a dose of “about 1 mg/kg-10mg/kg of selenium,” depending on the particular compound administered.

In a further example, when delivered topically, an exemplarytherapeutically effective dosage of a selenium-containing COX-2inhibitor described herein is in the range of about 0.1-2 ppm selenium,administered daily. In one example, topically administeredselenocoxib-1-GSH, selenocoxib-1-cysteine and/or selenocoxib-1-NAC isgiven at a dose of 1.5 ppm selenium (equivalent to 5.12 mg/kg bodyweight) every day.

In a further example, when delivered orally, an exemplarytherapeutically effective dosage of a selenium containing compounddescribed herein is in the range of about 1-15 ppm selenium. In aparticular example, an oral dose in the range of 1-15 ppm of seleniumequivalent to 3.412 mg/kg-51.18 mg/kg body weight of selenocoxib-1-GSHis a therapeutically effective dose.

A therapeutically effective amount of a pharmaceutical compositionaccording to the present invention will vary depending on the particularpharmaceutical composition used, the severity of the condition to betreated, the species of the subject, the age and sex of the subject andthe general physical characteristics of the subject to be treated. Oneof skill in the art could determine a therapeutically effective amountin view of these and other considerations typical in medical practice.In general it is contemplated that a therapeutically effective amountwould be in the range of about 0.001 mg/kg-100 mg/kg body weight,optionally in the range of about 0.01-10 mg/kg, and further optionallyin the range of about 0.1-5 mg/kg. Further, dosage may be adjusteddepending on whether treatment is to be acute or continuing.

Advantageously, selenium-containing COX-2 inhibitors according toaspects of the present invention are formulated to achievelipid-solubility and/or aqueous-solubility.

In particular aspects, a pharmaceutically acceptable carrier is aparticulate carrier such as lipid particles including liposomes,micelles, unilamellar or mulitlamellar vesicles; polymer particles suchas hydrogel particles, polyglycolic acid particles or polylactic acidparticles; inorganic particles such as calcium phosphate particles suchas described in for example U.S. Pat. No. 5,648,097; andinorganic/organic particulate carriers such as described for example inU.S. Pat. No. 6,630,486.

A particulate pharmaceutically acceptable carrier can be selected fromamong a lipid particle; a polymer particle; an inorganic particle; andan inorganic/organic particle. A mixture of particle types can also beincluded as a particulate pharmaceutically acceptable carrier.

A particulate carrier is typically formulated such that particles havean average particle size in the range of about 1 nm-10 microns. Inparticular aspects, a particulate carrier is a nanoparticulate carrierformulated such that particles have an average particle size in therange of about 1 nm-100 nm.

Selenium-containing COX-2 inhibitor compositions of the presentinvention optionally include a lipid-based carrier. The term“lipid-based carrier” refers to macromolecular structures having lipidand/or lipid derivatives as the major constituent.

Lipids included in lipid-based carriers can be naturally-occurringlipids, synthetic lipids or combinations thereof.

A lipid-based carrier is formulated as a liposome for use incompositions, kits and methods according to aspects of the invention.The term “liposome” refers to a bilayer particle of amphipathic lipidmolecules enclosing an aqueous interior space. Liposomes are typicallyproduced as small unilammellar vesicles (SUVs), large unilammellarvesicles (LUVs) or multilammellar vesicles (MLVs). A selenium-containingCOX-2 inhibitor composition of the present invention is associated withliposomes by encapsulation in the aqueous interior space of theliposomes, disposed in the lipid bilayer of the liposomes and/orassociated with the liposomes by binding, such as ionic binding orassociation by van der Waals forces. Thus, selenium-containing COX-2inhibitor composition of the present invention is contained in liposomeswhen it is encapsulated in the aqueous interior space of the liposomes,disposed in the lipid bilayer of the liposomes and/or associated withthe liposomes by binding, such as ionic binding or association by vander Waals forces. Liposomes according to aspects of the invention aregenerally in the range of about 1 nanometer-1 micron in diameteralthough they are not limited with regard to size. A particulate carrieris typically formulated such that particles have an average particlesize in the range of about 1 nm-10 microns. In particular aspects, aparticulate carrier is a nanoparticulate carrier formulated such thatparticles have an average particle size in the range of about 1 nm-100nm.

Liposomal formulations of selenium-containing COX-2 inhibitorcompositions according to aspects of the present invention include caninclude one or more types of neutral, cationic lipid and/or anioniclipid, such that the liposomal formulations have a net neutral surfacecharge at physiological pH. According to aspects, a PEG-modified lipidis included.

The term cationic lipid refers to any lipid which has a net positivecharge at physiological pH. Examples of cationic lipids include, but arenot limited to, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (DOTMA); 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);dioctadecylamidoglycylspermine (DOGS);1,2-dipalmitoylphosphatidylethanolamidospermine (DPPES);2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA); dimyristoyltrimethylammonium propane (DMTAP);(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium (DMRIE);dioctadecyldimethylammonium chloride (DODAC), Dimethyldidodecylammoniumbromide (DDAB); 3β[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol(DC-Chol);1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxyethyl)-imidazolinium(DOTIM); bis-guanidinium-spermidine-cholesterol (BGTC);bis-guanidinium-tren-cholesterol (BGTC);1,3-Di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER)N-[3-[2-(1,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethylammoniumiodide (YKS-220); as well as pharmaceutically acceptable salts andmixtures thereof. Additional examples of cationic lipids are describedin Lasic and Papahadjopoulos, Medical Applications of Liposomes,Elsevier, 1998; U.S. Pat. Nos. 4,897,355; 5,208,036; 5,264,618;5,279,833; 5,283,185; 5,334,761; 5,459,127; 5,736,392; 5,753,613;5,785,992; 6,376,248; 6,586,410; 6,733,777; and 7,145,039.

The term neutral lipid refers to any lipid which has no net charge,either uncharged or in neutral charge zwitterionic form, atphysiological pH. Examples of neutral lipids include, but are notlimited to, L-alpha-phosphatidylcholine (ePC),distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine(DOPE), distearoylphosphatidylethanolamine (DSPE);1,2-dioleoyl-sn-glycero-3-Phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), cephalin,ceramide, cerebrosides, cholesterol, diacylglycerols, and sphingomyelin.

The term anionic lipid refers to any lipid which has a net negativecharge at physiological pH. Examples of anionic lipids include, but arenot limited to, dihexadecylphosphate (DhP), phosphatidyl inositols,phosphatidyl serines, such as dimyristoyl phosphatidyl serine, anddipalmitoyl phosphatidyl serine., phosphatidyl glycerols, such asdimyristoylphosphatidyl glycerol, dioleoylphosphatidyl glycerol,dilauryloylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol,distearyloylphosphatidyl glycerol, phosphatidic acids, such asdimyristoyl phosphatic acid and dipalmitoyl phosphatic acid anddiphosphatidyl glycerol.

The term “modified lipid” refers to lipids modified to aid in, forexample, inhibiting aggregation and/or precipitation, inhibiting immuneresponse and/or improving half-life in circulation in vivo. Modifiedlipids include, but are not limited to, pegylated lipids, such aspolyethyleneglycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DPPE-PEG-2000), and polyethyleneglycol 750octadecylsphingosine (PEG(750) C8).

Exemplary ratios of components included in liposomal formulations of thepresent invention are neutral lipid:polyethyleneglycol modified neutrallipid—80:20 mol %.

For example, liposomal formulations include L-alpha-phosphatidylcholineand1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] in an 80:20 mol % ratio according to aspects of thepresent invention.

Thus, according to aspects of the present invention, liposomalformulations of selenium-containing COX-2 inhibitor compositions includeat least one polyethylene glycol modified neutral lipid, wherein thetotal amount of polyethylene glycol modified neutral lipid is an amountin the range of 10-30 molar percent, inclusive, such as 15-25 molarpercent polyethylene glycol modified neutral lipid and further includinganionic, cationic or neutral lipids, with the proviso that the resultingliposomes have a net neutral surface charge at physiological pH.

According to aspects of the present invention, liposomal formulations ofselenium-containing COX-2 inhibitor compositions have an averageparticle size in the range of 1-100 nm, a neutral or near neutral chargein normal saline and are characterized by stability in normal saline at4° C. for at least 1-12 months and at 20° C. for at least one month.

Aspects of liposomal formulations of the present invention include aliposomal formulation of selenocoxib-1-GSH with various ratios ofePC:DPPE:PEG-2000. Exemplary ratios of components included in liposomalformulations of the present invention are neutral lipid ePC:DPPE-PEG-2000 in a ratio of 80:20 mol %.

In addition to containing one or more selenium-containing COX-2inhibitor compositions of the present invention, liposomes of thepresent invention optionally contain any of a variety of usefulbiologically active molecules and substances including, but not limitedto, one or more additional therapeutic agents, proteins, peptides,carbohydrates, oligosaccharides, drugs, and nucleic acids capable ofbeing complexed with the liposomes. The term “biologically activemolecules and substances” refers molecules or substances that exert abiological effect in vitro and/or in vivo, such as, but not limited to,nucleic acids, inhibitory RNA, siRNA, shRNA, ribozymes, antisensenucleic acids, antibodies, hormones, small molecules, aptamers, decoymolecules and toxins.

Liposomes are generated using well-known standard methods, including,but not limited to, solvent/hydration methods, ethanol or etherinjection methods, freeze/thaw methods, sonication methods,reverse-phase evaporation methods, and surfactant methods. Liposomes andmethods relating to their preparation and use are found in Liposomes: APractical Approach (The Practical Approach Series, 264), V. P. Torchilinand V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003; N.Duzgunes, Liposomes, Part A, Volume 367 (Methods in Enzymology) AcademicPress; 1st ed., 2003; L. V. Allen, Jr. et al., Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, Pa.:Lippincott, Williams & Wilkins, 2005, pp. 663-666; and A. R. Gennaro,Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins, 21st ed., 2005, pp. 766-767.

A pharmaceutical composition includes a liposomal formulation ofselenocoxib-1-GSH in particular aspects of the present invention.

A pharmaceutical composition includes a liposomal formulation ofselenocoxib-1-NAC in particular aspects of the present invention.

A pharmaceutical composition includes a liposomal formulation ofselenocoxib-1-cysteine in particular aspects of the present invention.

Liposomal formulations of selenium-containing COX-2 inhibitorcompositions of the present invention are administered by anyappropriate route including, but not limited to, intravenous injectionand/or topical application according to aspects of the presentinvention.

Commercial Packages

Commercial packages are provided according to aspects of the presentinvention for treating cancer in a subject in need thereof, including aselenium-containing COX-2 inhibitor; selenocoxib-1-GSH,selenocoxib-1-cysteine, selenocoxib-1-N-acetylcysteine; or apharmaceutically acceptable salt, ester, amide, solvate or stereoisomerthereof. One or more auxiliary components are optionally included incommercial packages of the present invention, such as a pharmaceuticallyacceptable carrier exemplified by a buffer, diluent or a reconstitutingagent.

Aspects of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Cell Lines and Culture Conditions

Normal human primary melanocytes containing wild-type B-Raf FOM103 andNHEM 558 are cultured as described in Satyamoorthy K et al., MelanomaRes 1997, 7 Suppl 2:S35-42. Human fibroblast FF2441 cells, metastaticmelanoma cell lines harboring mutant ^(V600E)B-Raf: UACC 903, A375M(ATCC CRL-1619) and 1205 Lu are maintained in DMEM (Invitrogen),supplemented with 10% FBS (Hyclone). Radial (WM35 harboring mutant^(V600E)B-Raf) and vertical (WM115, WM278.1 both harboring mutant^(V600E) B-Raf) growth phase melanoma cell lines are maintained in Tu2%medium as described in Quong R Y et al., Melanoma Res 1994, 4:313-9.Wild type B-Raf containing C8161.C19 and MelJuSo cell lines weremaintained in DMEM supplemented with 10% FBS. Cell lines are maintainedin a 37° C. humidified 5% CO₂ atmosphere incubator and periodicallymonitored for genotypic characteristics, tumorigenic potential toconfirm cell line identity and phenotypic behavior.

Statistical Analysis

Statistical analysis is performed using Prism 4.0 GraphPad Software.One-way or Two-way Analysis Of Variance (ANOVA) is used for group wisecomparisons, followed by the Tukey's or Bonferroni's post hoc tests. Forcomparison between two groups, the t test is used. Results represent atleast two to three independent experiments and are shown asaverages±S.E.M. Results with a P value less than 0.05 (95% CI) areconsidered significant.

Western Blot Analysis

Cell lysates are harvested and processed as described in Sharma A etal., Cancer Res 2006, 66:8200-9. Treatment conditions: 1.5×10⁶ melanomacells are plated in 100 mm culture dishes, 48 h later, treated withcelecoxib, selenocoxib-1-GSH (5-20 μmol), PLX-4032 (0.2-20 μmol/L) orU0126 (2.5-50 μmol/L) for 6 to 72 h. Protein lysates are collected forWestern blotting. Blots are probed with total and phospho-Akt (Ser473),phospho-PRAS40 (Thr246), phospho-Erk1/2 (Thr202/Tyr204), total andPhospho-Mek1/2 (Ser217) and cleaved PARP from Cell Signaling Technology(Danvers, Mass.). Total PRAS40 from Invitrogen (Carlsbad, Calif.). TotalErk, cyclin D1, p27, Alpha-enolase and secondary antibodies conjugatedwith horseradish peroxidase from Santa Cruz Biotechnology (Santa CruzBiotechnology, Santa Cruz, Calif.). COX-1 and COX-2 antibodies wereobtained from Cayman Chemical Company, Ann Arbor, Mich. Immunoblots aredeveloped using the enhanced chemiluminescence detection system(Amersham Pharmacia Biotech, Piscataway, N.J.).

COX-2 expression is elevated in advanced melanoma patient tumors andmelanoma cell lines.

The expression of COX-2 is measured by Western blotting in a panel ofmelanoma patient tumors and cell lines representing radial (WM35),vertical (WM115, WM278.1) and metastatic (A375M, UACC 903, 1205 Lu)stages of development. For analysis of human melanoma tumors, tissuesamples are collected from 25 patients at surgery, immediately snapfrozen in liquid nitrogen, and stored at −80° C. until protein lysatecollection. Tumors are pulverized using a mortar and pestle chilled inliquid nitrogen to collect protein for Western blotting. Protein lysatesare extracted from tumors as described in Madhunapantula S V et al.,Cancer Res 2007, 67:3626-36, and analyzed by Western blotting to measurelevels of COX-2. COX-2 protein levels in tumors are normalized to anAlpha-enolase loading control and relative COX-2 expression quantifiedusing ImageJ software.

FIG. 1A shows elevated levels of COX-2 expression in melanoma patienttumors and cell lines. Results are normalized to Alpha-enolase loadingand compared to normal human melanocyte controls. 76% (19/25) ofmelanoma patient tumors had elevated COX-2 expression when compared tonormal human melanocyte control (NHEM) cells.

All melanoma cell lines examined expressed levels of COX-2 proteinhigher than that observed in melanocytes, albeit, in varying amounts.FIG. 1B shows that COX-2 expression is increased in a cell line basedmelanoma tumor progression model. Lysates collected from normal humanmelanocytes, radial (WM35), vertical (WM115, WM278.1), and metastatic(A375M, UACC and 1205 Lu) stage cell lines are subjected to Western blotanalysis and probed for COX-2. Alpha-enolase serves as a control forequal protein loading. Expression of COX-2 in UACC 903, A375M and 1205Lu cell lines is 43, 76 and 329-fold higher respectively, than thatobserved in melanocytes.

SiRNA Efficacy and Knockdown Studies:

To determine efficacy of siRNA-mediated knockdown, 200 pmoles of siCOX-2#1 or siCOX-2 #2, is compared to scrambled siRNA or reconstitutionbuffer following nucleofection into 1×10⁶ of 1205 Lu and A375M cellsusing an Amaxa nucleofector with solution R/program K-17 (1205 Lu) orA-23 (A375M). Transfection efficiency of viable cells is 70-80%.Following siRNA transfection, cells recovered for 2 days and arereplated in 96-well plates to measure cell viability using the MTS assay(CellTiter 96 AQueous Cell Proliferation Assay, Promega, Madison, Wis.).To show siRNA-mediated protein knockdown in vitro, 1×10⁶ of 1205 Lu,UACC 903 (Program K-17) and A375M cells are similarly nucleofected with200 pmoles of siCOX-2 #1, siCOX-2 #2, and 100 pmoles of ^(V600E)B-RAF,MEK1, MEK2, ERK1, and ERK2, scrambled siRNA, reconstitution buffer, andprotein lysates are harvested at day 4 or 6, and analyzed by Westernblot analysis. Duplexed Stealth siRNA (Invitrogen, Carlsbad, Calif.) isused for these studies. The following siRNA sequences are used: COX-2#1: UCC AGA CAA GCA GGC UAA UAC UGA U (SEQ ID NO: 1); COX-2 #2: GAG UUAUGU CUU GAC AUC CAG AUC A (SEQ ID NO: 2). SiRNA sequences for scrambled,^(V600E)B-RAF, MEK1, MEK2, ERK1, and ERK2 as described in Sharma A etal., Cancer Res, 2006, 66:8200-9.

Reduction of COX-2 protein levels using siRNA targeting ^(V600E)B-Raf orCOX-2 decreased melanoma cell viability. To determine whether targetingCOX-2 would reduce viability of melanomas, metastatic 1205 Lu and A375Mcells that express relatively high levels of protein are transfectedwith 2 different siRNAs targeting different regions of the mRNA and cellviability compared to controls nucleofected with a scrambled siRNA orbuffer. FIG. 1C shows that targeting COX-2 using siRNAs decreasedmelanoma cell viability. 1205 Lu and A375M melanoma cells arenucleofected with two non-overlapping siRNAs against COX #1 and #2 usingsolution R/program K-17 (1205 Lu) or solution R/program A-23 (A375M).Targeting COX-2 reduced melanoma viability by 32 to 63%. siRNA targetingmutant ^(V600E)B-Raf serves as a positive control. Alpha-enolase servesas a control for equal protein loading.

In both cell lines, targeting COX-2 reduced melanoma viability by 32 to63%. Targeting mutant ^(V600E)B-Raf using siRNA reduced COX-2expression, suggesting that protein expression is regulated through thispathway, FIG. 1C. Levels of COX-2 protein decreased when ^(V600E)B-Rafexpression levels are targeted using siRNA.

SiRNA and pharmacological agents targeting the ^(V600E)B-Raf confirmthat COX-2 expression is regulated through ^(V600E)B-Raf signalingpathway in melanomas. To examine whether siRNA-mediated targeting ofMek1/2 and Erk1/2, downstream of ^(V600E)B-Raf would decrease COX-2expression, siRNA or pharmacological agents are used to down regulateprotein expression or activity. 1205 Lu and UACC 903 cells arenucleofected with siRNAs inhibiting mutant ^(V600E)B-Raf, downstreamMek1/2 or Erk1/2. A significant decrease in COX-2, but not the COX-1isoform, is observed when each of the members of the ^(V600E)B-Rafsignaling pathway is targeted. FIG. 1D shows that SiRNA mediatedinhibition of the MAP kinase pathway decreased COX-2 expression inmelanomas. 1205 Lu and UACC 903 cells are nucleofected with siRNAsinhibiting mutant ^(V600E)B-Raf, Mek1 or Mek2 and Erk1 or Erk2. Comparedto cells treated with scrambled siRNA, decreasing protein levels of eachmember of the MAP kinase pathway led to a decrease in COX-2 but notCOX-1 levels. Alpha-enolase serves as a control for equal proteinloading.

Vemurafenib (PLX-4032), a ^(V600E)B-Raf inhibitor, is used to inhibitactivity of this pathway. Cells treated with PLX-4032 showed decreasedCOX-2 protein expression beginning after 12 h of treatment. The changesare more evident following 12, 24 and 48 h of treatment in the case of1205 Lu, FIG. 1E. In the case of UACC 903 cells, a significant decreaseis seen after 24 and 48 h of treatment, FIG. 1F. Similar to siRNAstudies, no changes are observed in COX-1 expression in either cell lineunder these treatment conditions, FIGS. 1C and 1D. A decrease inphosphorylation, but not expression of Mek1/2 and Erk1/2 proteins showedthe inhibitory activity of PLX-4032 on the ^(V600E)B-Raf pathway. FIGS.1E-F show that PLX-4032 targeting of ^(V600E)B-Raf decreased COX-2expression. 1205 Lu or UACC 903 cells are treated with 0.2-20 μmol/LPLX-4032 for 6, 12, 24 and 48 h. Levels of pMek1/2, pErk1/2, and COX-2decreased following after 12 h of drug treatment. No changes are seen inCOX-1 expression. Alpha-enolase serves as a control for equal proteinloading.

Like PLX-4032, Mek1/2 inhibitor U0126 also reduces the expression ofCOX-2 without affecting COX-1 in 1205 Lu and UACC 903 cell lines. FIG.1G shows that U0126 targeting of Mek1/2 decreased COX-2 expression. 1205Lu or UACC 903 cells are treated with 2.5-50 μmol/L U0126 for 48 h.Levels of pErk1/2 and COX-2 similarly decrease following drug treatmentwith no changes in COX-1 expression. Alpha-enolase serves as a controlfor equal protein loading.

Targeting ^(V600E)B-Raf or downstream proteins in the signaling cascadereduces expression of COX-2 in melanomas and decreases the proliferativepotential of the cells. Thus, COX-2 lies downstream of ^(V600E)B-Raf,Mek-1/2 and Erk-1/2 in this signaling pathway.

Synthesis of Celecoxib, Selenocoxib-1 and Selenocoxib-1-GSH:

Celecoxib is synthesized as described in detail in Penning T D et al. JMed Chem 1997; 40: 1347-65. Selenocoxib-1 is prepared as described inDesai D et al., Chem Biol Interact 2010; 188: 446-56.

Selenocoxib-1-GSH is prepared by reacting molar equivalent selenocoxib-1with glutathione in tetrahydrofuran: H₂O (2:1) mixture. pH is adjustedto slightly basic to give selenocoxib-1-GSH conjugate in quantitativeyield as yellow powder. MP: 196-198° C.; ¹H NMR (DMSO-d₆, 500 MHz) d1.72-1.83 (m, 3H), 1.88-1.98 (m, 1H), 2.23-2.36 (m, 2H), 2.91 and 2.94(dd, 1H, J=10 Hz), 3.16 and 3.18 (dd, 1H, J=4.5 Hz), 3.57-3.72 (m, 3H),4.18 and 4.23 (dd, 2H, J=12.5 Hz and 22 Hz), 4.51 (td, 1H, J=4.0 Hz),6.62 (s, 1H, CH), 7.25 (d, 1H, aromatic, J=2.5 Hz), 7.26 (d, 1H,aromatic, J=3.5 Hz), 7.35-7.39 (m, 3H, aromatic), 7.41 (dt, 2H,aromatic, J=8.5 Hz and 2.0 Hz), 7.79 (dt, 2H, aromatic, J=8.5 Hz and 2.0Hz), 8.44 (d, 1H, J=7 Hz), 8.75 (ds, 1 H); MS (M/Z, Intensity): 681 (M+,100). The identities of the compounds are confirmed by nuclear magneticresonance as well as mass spectra analysis, and purity (>99%) isquantified by high-performance liquid chromatography analysis.

Selenocoxib-1-NAC Preparation

To a solution of selenocoxib-1 (2.0 mmol) in THF (25 mL) at 0° C. isadded a solution of N-acetylcysteine (326 mg, 2.0 mmol) in water (15mL), and to this mixture 2% NaOH (0.1 mL) is added. The reaction mixtureis allowed to warm to room temperature and stirred for 12 h. The mixtureis washed with hexane (2×5 mL), and the aqueous layer is acidified with2 N HCl and extracted with ethyl acetate. The organic layer isseparated, dried (MgSO₄), and concentrated in vacuum to giveselenocoxib-1-NAC in quantitative yield.

Selenocoxib-1-Cysteine Preparation

To a solution of selenocoxib-1 (2.0 mmol) in THF (25 mL) at 0° C. isadded a solution of cysteine (326 mg, 2.0 mmol) in water (15 mL), and tothis mixture 2% NaOH (0.1 mL) is added. The reaction mixture is allowedto warm to room temperature and stirred for 12 h. The mixture is washedwith hexane (2×5 mL), and the aqueous layer is acidified with 2 N HCland extracted with ethyl acetate. The organic layer is separated, dried(MgSO₄), and concentrated in vacuum to give selenocoxib-1-cysteine inquantitative yield.

Unexpectedly, selenocoxib-1-GSH has significantly lower toxicity onnormal cells and has melanoma cell killing efficacy, FIG. 2B.Selenocoxib-1 inhibited melanoma cell viability and also reduces normalcell growth with IC₅₀s similar to that of melanoma cells, indicatingtoxicity. Surprisingly, selenocoxib-1-GSH has an increased differentialinhibition ratio, that is, the number of inhibited normal cells issignificantly reduced compared to the number of inhibited cancer cellswhen both are treated with selenocoxib-1-GSH, FIG. 2B.Selenocoxib-1-GSH, but not selenocoxib-1, inhibits the growth ofmelanoma cells with a lesser effect on the growth of normal humanmelanocytes or fibroblast cells. Similar results are expected withselenocoxib-1-cysteine and selenocoxib-N-acetylcysteine.

Toxicity of selenocoxib-1 but not selenocoxib-1-GSH is observed inanimal studies. Animal survival and body weight loss following treatmentwith celecoxib, selenocoxib-1 or selenocoxib-1-GSH. Celecoxib (0.127μmoles), selenocoxib-1 (0.032-0.064 moles) or selenocoxib-1-GSH(0.127-0.254 μmoles) are injected intraperitoneally daily for seven days(3 mice/group). Number of mice surviving and changes in body weightafter 7 days of drug treatment is scored (Table I).

Celecoxib at 0.127 μmoles led to death of all animals following 7 daysof treatment, while selenocoxib-1 at concentrations of 0.032-0.064μmoles, (equivalent to ppm selenium) leads to weight losses of 14% or100% animal mortality after seven days of treatment (Table I). Incontrast, animals receiving 0.127 to 0.254 μmoles (equivalent to 5 to 10ppm of selenium) of selenocoxib-1-GSH exhibit negligible weight loss of˜2% and no mortality is observed. These data are summarized in Table I.

TABLE I Doses (μmoles, equivalent to Doses Mortality % of weightCompounds ppm selenium) (PPM) at Day 7 loss Celecoxib 0.127 — 3/3 —Selenocoxib-1 0.032 2.5 0/3 14.07 0.064 5.0 3/3 — Selenocoxib-1-GSH0.127 10 0/3 No loss 0.254 20 0/3 2.0

Cyclooxygenase Inhibition

Human recombinant COX-2 activity is assayed using a commercialCOX-inhibitor screening assay kit (Cayman Chemical, Ann Arbor, Mich.)and following the manufacturer's protocol. The concentrations ofcelecoxib and selenocoxib-1-GSH tested are 0.2, 2.0 and 20 nM. SC-560and DuP-697, standard inhibitors for COX-1 and COX-2 respectively, aretested as positive controls. DMSO serves as the negative controls (100%activity). The product of this enzymatic reaction is determinedspectrophotometrically at 405 nm. The optical activity is proportionalto the amount of PG-acetylcholinesterase conjugate bound to the well,which is inversely proportional to the amount of PGs present in the wellduring incubation. This assay is performed in duplicate.

FIG. 2C shows that selenocoxib-1-GSH has COX-2 inhibitory activity.Human recombinant COX-2 activity is assayed using a commercialCOX-inhibitor screening assay kit. 0.2, 2.0 and 20 nM of celecoxib andselenocoxib-1-GSH of are tested and shown to retain COX-2 inhibitoryactivity. DMSO serves as the negative controls (100% activity). Theproduct of this enzymatic reaction is determined spectrophotometricallyat 405 nm. Assay is performed in 2 independent experiments.

Cell viability, proliferation, apoptosis and cell cycle analysis:Viability and IC₅₀ (μmol/L) of normal human melanocytes, fibroblast andmelanoma cells following treatment with inhibitors are measured usingthe MTS assay (Promega, Madison, Wis.) described in Sharma A, et al.,Clin Cancer Res 2009; 15:1674-85; and Madhunapantula S V, et al., MolCancer Ther 2008; 7:1297-308. In brief, 5×10³ cells per well in 100 ptof media are plated and grown in a 96-well plate for 36 to 72 hrespectively for melanoma (WM35, WM115, 1205 Lu and UACC 903) and normalcell lines (FOM103 and FF2441) treated 0.312 to 100 μmol/L of celecoxib,selenocoxib-1 and selenocoxib-1-GSH for 24, 48 or 72 h with DMSO asvehicle control. IC₅₀ values for each inhibitor in μmol/L for respectivecell lines are measured from three independent experiments usingGraphPad Prism version 4.01 (GraphPad Software, La Jolla, Calif.).

FIGS. 3A, 3B & 3C show that selenocoxib-1-GSH, but not celecoxib,inhibited melanoma cell proliferation and induced apoptosis. 1205 Lu andUACC 903 cells are treated with increasing concentrations of celecoxiband selenocoxib-1-GSH for 72 h and cell viability, proliferation andapoptosis rates measured by MTS, BrdU incorporation and caspase-3/7assays, respectively. Data represent averages of at least 3 independentexperiments; bars; S.E.M.

Dose response curves generated using 1205 Lu and UACC 903 cell linesdemonstrate that selenocoxib-1-GSH but not celecoxib inhibits melanomacell viability, FIG. 3A. At 12.5 μmol/L, selenocoxib-1-GSH leads to a 40to 60% decrease in cell viability compared to control DMSO vehicletreated cells, FIG. 3A. Furthermore, selenocoxib-1-GSH inhibited thegrowth of melanoma cell lines irrespective of B-Raf mutation status.

Table II shows that selenocoxib-1-GSH kills melanoma cells moreeffectively than normal cells. Normal and melanoma cells are seeded into a 96-well plate and after 36 to 72 h, treated with increasingconcentrations of celecoxib, selenocoxib-1 or selenocoxib-1-GSH for theindicated time period. The number of viable cells is measured using MTSand percentage decrease in viability calculated. IC₅₀ values for eachinhibitor in μmol/L for respective cell lines are measured from threeindependent experiments using GraphPad Prism version 4.01 (GraphPadSoftware, La Jolla, Calif.).

TABLE II FOM103 FF2441 WM35 WM115 UACC 903 1205 Lu Celecoxib >100 >10051.3 ± 2.3 54.4 ± 3.6 >100 >100 24 h Selenocoxib-1-GSH 66.3 ± 3.0 >10052.6 ± 3.8 30.1 ± 3.3 30.9 ± 2.8 24.6 ± 2.2 Celecoxib >100 >100 42.3 ±1.8 45.3 ± 2.7 >100 83.6 ± 3.3 48 h Selenocoxib-1-GSH 53.4 ± 4.3 75.5 ±5.6  4.1 ± 0.8  5.8 ± 0.9 20.6 ± 1.7 17.2 ± 1.6 Celecoxib 68.0 ± 1.265.3 ± 3.3 37.9 ± 3.3 41.8 ± 2.9 76.6 ± 4.4 66.1 ± 3.2 72 hSelenocoxib-1-GSH 41.2 ± 3.2 36.3 ± 5.6  2.7 ± 0.4  3.1 ± 0.4 14.2 ± 1.210.6 ± 2.6 Normal Radial Vertical Metastatic

Mechanisms leading to cell growth inhibition after treatment withselenocoxib-1-GSH are examined by measuring the level of cellproliferation, apoptosis, and the percentage of cells in the variousphases of the cell cycle.

Cellular proliferation and apoptosis rates are measured by seeding 5×10³cells in 96-well plates, followed by treatment for 72 h with celecoxiband selenocoxib-1-GSH. Percentage of proliferating and apoptotic cellsare quantified by a colorimetric using cell proliferation ELISA BrdU kit(Roche Applied Sciences, Indianapolis, Ind.) and Apo-ONE Homogenouscaspase-3/7 assay kit (Promega, Madison Wis.), as described in Sharma A,et al., Clin Cancer Res 2009; 15:1674-85; and Madhunapantula S V, etal., Mol Cancer Ther 2008; 7:1297-308.

Selenocoxib-1-GSH reduces proliferation of 1205 Lu and UACC 903 melanomacells, FIG. 3B, and increases caspase-3/7 activity, which is anindicator of apoptosis, in a dose dependent manner up to 25 μmol/L, FIG.3C. A significant decrease in caspase-3/7 activity is observed when UACC903 cells are treated with 50 μmol/L selenocoxib-1-GSH, which can beattributed to massive cell death caused by degradation of cellularproteins, FIG. 3C.

The effect of selenocoxib-1-GSH on cell cycle distribution is measuredby analyzing propidium iodide stained 1205 Lu and UACC 903 cells using aBD FACScan. Melanoma cells 1205 Lu and UACC 903 are grown in 100-mmculture dishes followed by treatment with 12.5 and 25 μmol/L ofcelecoxib and selenocoxib-1-GSH for 72 h (FIG. 3D). Total floating andadherent cells are collected following trypsinization and stained usinga 1 mL propidium iodide solution containing 100 μg/mL propidium iodide;(Sigma, St Louis, Mo.), 20 μg/mL Ribonuclease A (Roche diagnostics,Indianapolis, Ind.) and 3 μg/mL Triton X-100 dissolved in 0.1% (W/V)sodium citrate for 30 m at 4° C. Stained cells are analyzed using theFACScan analyzer (Becton Dickinson, Franklin lakes, NJ) and dataprocessed utilizing ModFit LT software (Verity Software House, Topsham,Me.) as described in Sharma A, et al., Clin Cancer Res 2009; 15:1674-85;and Madhunapantula S V, et al., Mal Cancer Ther 2008; 7:1297-308.

Selenocoxib-1-GSH treatment increases the sub-G₀-G₁ cell population,which is indicative of cellular apoptosis. FIG. 3D shows thatselenocoxib-1-GSH arrested melanoma cells in the G₀-G₁ phase of the cellcycle. 1205 Lu and UACC 903 cells are treated with 12.5 and 25 μmol/L ofcelecoxib, selenocoxib-1-GSH or vehicle DMSO control for 72 h. Totalfloating and adherent cells are collected, and stained with propidiumiodide to analyze the distribution of cells in different phases of thecell cycle stages using a FACScan analyzer. Selenocoxib-1-GSH, but notcelecoxib treatment, inhibits cell cycle progression by increasing thesub-G₀-G₁ population and arresting cells in the G₀-G₁ phases of the cellcycle. Data represents an average of 2 independent experiments.

The number of sub-G₀-G₁ population cells increases by 6.2 and 4.7-fold,respectively when 1205 Lu and UACC 903 cells are treated with 25 μmol/Lselenocoxib-1-GSH (FIG. 3D). In addition, increase in the G₀-G₁ cellpopulation is also observed with 12.5 and/or 25 μmol/L selenocoxib-1-GSHtreatment in these cell lines (FIG. 3D). Thus, selenocoxib-1-GSHinhibits cellular proliferation and triggered apoptosis mediated througha G₀-G₁ block, resulting in fewer cells in S and G₂-M phases.

Animal Studies Using siRNA Tumorigenicity Assessments:

Tumor kinetic studies are carried out in athymic-Foxn1^(nu) nude mice(Harlan Sprague Dawley, Ind.). 200 pmoles of siRNA COX-2 #1 or COX-2 #2are nucleofected into 2×10⁶ 1205 Lu cells and after 48 h of recovery,1×10⁶ cells are collected in 0.2 mL of 10% FBS-DMEM and injectedsubcutaneously above both the left and right rib cages of 4-6 week oldfemale mice (5 mice/group). Dimensions of developing tumors are measuredon alternate days up to day 21.5, using calipers by L×W×D (mm³) asdescribed in Sharma A, et al., Clin Cancer Res 2009; 15:1674-85.

siRNA targeting COX-2 is used to reduce protein expression in melanomacells to measure the effect on melanoma tumor development. 1205 Lumelanoma cells are nucleofected with siRNA targeting COX-2. 48 h later,viable cells are subcutaneously injected into left and right flanks ofnude mice. Developing tumors are measured on alternate days for 21.5days (FIG. 4A). The tumorigenic potential of COX-2 treated cellsdecreased by ˜70% compared to buffer or scrambled siRNA control cells.Inhibition of COX-2 protein expression using siRNAs, reduces xenograftedmelanoma tumor development by an average of 71% after 21 days comparedto control buffer and siScrambled treated tumors. FIG. 4A shows thatsiRNA-mediated targeting of COX-2 decreases melanoma tumor developmentin mice.

Selenocoxib-1-GSH inhibits melanoma tumor development in mice withoutsignificant toxicity.

Tumor kinetics are measured by subcutaneous injection of 1×10⁶ 1205 Luor UACC 903 cells in 0.2 mL of DMEM supplemented with 10% FBSsubcutaneously injected above both left and right rib cages of 3- to4-wk-old female athymic-Foxn1″ nude mice (Harlan Sprague Dawley, Ind.).Six days later, when a fully vascularized tumor (50-75 mm³) had formed,mice are randomly divided into DMSO vehicle control and experimentalgroups (5 mice/group; 2 tumors/mouse) and treated intraperitoneally withselenocoxib-1-GSH (0.127 μmoles, equivalent to 10 ppm selenium) orcelecoxib (0.127 μmoles) in DMSO on alternate days for 4 weeks. Bodyweight (grams) and dimensions of the developing tumors (mm³) aremeasured at the time of drug treatment as described in Sharma A, et al.,Clin Cancer Res 2009; 15:1674-85; and Madhunapantula S V, et al., MolCancer Ther 2008; 7:1297-308.

FIGS. 4B and 4C show results of this in vivo analysis. Selenocoxib-1-GSHreduces tumor development by 70.5% and 52% in 1205 Lu and UACC 903 cellsrespectively, compared to DMSO control treated mice (P<0.001; two-wayANOVA). No significant difference is observed in body weight of micetreated with the drug indicating negligible toxicity (FIGS. 4B and 4C;inset).

A significant decrease in xenograft tumor development compared tovehicle DMSO treated mice is observed from day 16 in 1205 Lu tumors,FIG. 4B. Similarly, a significant decrease is also observed in UACC 903tumors from day 22, FIG. 4C. For both cell lines at the end oftreatment, up to a 70% decrease in tumor volume is observed followingselenocoxib-1-GSH treatment compared to controls, FIGS. 4B and 4C. Nonoticeable changes in animal body weight are observed, FIGS. 4B and 4C;inset.

Toxicity Assessments

Four to six week old athymic-Foxn1^(nu) nude mice (Harlan SpragueDawley, Ind.), are treated with either vehicle control orselenocoxib-1-GSH (n=5) as described in tumor kinetics studies. At theend of treatment, blood is collected from each sacrificed animal in aplasma separator tube with lithium heparin (BD Microtainer) followingcardiac puncture and analyzed for ALKP (Alkaline phosphatase), ALT(Alanine aminotransferase), AST (Aspartate aminotransferase), ALB (Totalalbumin), TBIL (Total bilirubin), CREA (Creatinine), BUN (Blood ureanitrogen), CHOL (Total cholesterol), TRIG (Total triglyceride) and GLU(Glucose) levels to ascertain possible liver, heart, kidney, andpancreas related toxicity. A portion of vital organs, liver, heart,kidney, pancreas, and spleen-from each animal is formalin-fixed andparaffin-embedded to examine toxicity-associated changes in cellmorphology and tissue organization following H&E staining as describedin Nguyen N, et al. Cancer Prev Res (Phila) 2011; 4: 248-58.

The levels of blood markers for major organ related toxicity, ALKP(Alkaline phosphatase), ALT (Alanine aminotransferase), AST (Aspartateaminotransferase), ALB (Total albumin), TBIL (Total bilirubin), CREA(Creatinine), BUN (Blood urea nitrogen), CHOL (Total cholesterol), TRIG(Total triglyceride) and GLU (Glucose) are evaluated and levels indicatenegligible differences compared to controls at the concentrationsexamined. FIG. 4D shows that selenocoxib-1-GSH does not affect bloodbiomarkers indicative of major organ related toxicity. The levels ofblood markers for major organ related toxicity, ALKP (Alkalinephosphatase), ALT (Alanine aminotransferase), AST (Aspartateaminotransferase), ALB (Total albumin), TBIL (Total bilirubin), CREA(Creatinine), BUN (Blood urea nitrogen), CHOL (Total cholesterol), TRIG(Total triglyceride) and GLU (Glucose) are evaluated and levels indicatenegligible differences compared to controls at the concentrationsexamined.

Analysis of H&E stained tissue sections from control DMSO vehicle orselenocoxib-1-GSH treated mice show no changes in the morphology orarchitecture of the liver, heart, lung, kidney or spleen. These data, inFIG. 5, demonstrate that selenocoxib-1-GSH effectively inhibits melanomatumor development leading to tumor regression without significant organrelated toxicity.

Signaling Pathways

Selenocoxib-1-GSH inhibits Akt signaling, which activates MAP kinaseactivity to reduce melanoma cellular proliferation and promoteapoptosis.

pAkt levels are examined in 1205 Lu and UACC 903 melanoma cellsfollowing treatment. Compared to celecoxib, selenocoxib-1-GSH treatmentinhibited the Akt phosphorylation in a dose dependent manner (FIG. 6A).FIG. 6A shows that selenocoxib-1-GSH inhibits the PI3K/Akt signalingpathway. 1205 Lu and UACC 903 cells are treated with increasingconcentrations of celecoxib and selenocoxib-1-GSH for 72 h and celllysates analyzed to determine expression as well as activity of Aktsignaling proteins. Selenocoxib-1-GSH reduces expression ofphosphorylated Akt, pPRAS40. Alpha-enolase serves as a control for equalprotein loading.

Furthermore, phosphorylation of the downstream Akt3 substrate PRAS40 wassignificantly inhibited, FIG. 6A

1205 Lu and UACC 903 melanoma cells have elevated MAPK activities due topresence of constitutively active ^(V600E)B-Raf. Akt3 phosphorylates^(V600E)B-Raf to lower MAP kinase pathways activity to levels promotingrather than inhibiting cellular proliferation. FIG. 6B shows thatselenocoxib-1-GSH activates the MAPK signaling pathway.Selenocoxib-1-GSH treatment at 15 and 20 μmol/L in both 1205 Lu and UACC903 cell lines increased pErk1/2 levels. Alpha-enolase serves as acontrol for equal protein loading. Therefore, selenocoxib-1-GSHtreatment at 15 and 20 μmol/L concentrations in both 1205 Lu and UACC903 cell lines leads to higher pERK1/2 levels, FIG. 6B, due to decreasedphosphorylation and regulation of ^(V600E)B-Raf by Akt (Cheung et al.,Cancer Res. 68:3429-3439, 2008). In addition, selenocoxib-1-GSH alsoinhibits the expression of cyclin-D1 and increases the levels of p27,FIG. 6C. FIG. 6C shows that selenocoxib-1-GSH inhibits proteinindicating cell proliferation. Cyclin D1 levels decreased with aconcomitant increase in p27 following treatment with selenocoib-1-GSH.Alpha-enolase serves as a control for equal protein loading.

Finally, increased caspase 3/7 and cleaved PARP levels are observedindicating higher levels of apoptosis in selenocoxib-1-GSH compared tocelecoxib treated cells, FIG. 6D. FIG. 6D shows that selenocoxib-1-GSHincreased protein cleavage indicating apoptosis. Selenocoxib-1-GSHincreases caspase-3/7 activity and elevates levels of cleaved PARPprotein in melanoma cells. Alpha-enolase serves as a control for equalprotein loading.

Reactive Oxygen Species (ROS) Assay

Intracellular ROS are monitored according to a procedure described indetail in Smasundaram et al., Cancer Res., 62:3868-3875, 2002. 1.5×10⁶melanoma cells are plated in 100 mm culture dishes and 48 hours latertreated with 5-20 micromoles/liter concentration of celecoxib,selenocoxib-1 or selenocoxib-1-GSH. After 24 hours treatment, totalcells (floating and adherent) are collected in ice-coldphosphate-buffered saline and 5×10³ cells/well placed in 100 microlitersculture medium in a 96-well plate containing 10 micromoles/liter2′,7′-dichlorfluorescein-diacetate (commercially available from Sigma,St. Louis, Mo.) and incubated at 37° C. for 30 minutes. The amount ofROS present compared to DMSO vehicle treated cells was represented inarbitrary units. The assay is performed twice with four replicates eachtime, results are shown in FIGS. 7A and 7B.

Selenocoxib-1-GSH inhibits the generation of ROS. UACC 903 and 1205 Lucells were exposed to celecoxib, selenocoxib-1 and selenocoxib-1-GSH at5, 10, 15 and 20 μmol/L. Generation of ROS was assayed usingdichlorodihydrofluorescein diacetate (DCFH-DA) and fluorescence measuredat an emission wavelength of 530 nm and excitation wavelength of 485 nm.The GSH form of selenocoxib-1 more dramatically decreased ROS than theforms lacking it (FIGS. 7A and 7B).

Synthesis of a Liposomal Formulation of Selenocoxib-1-GSH

Selenocoxib-1-GSH can be encapsulated into nanoliposomes by combiningL-a-phosphatidylcholine (ePC) and1,2-dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (DPPE-PEG-2000) in chloroform at 80:20 mol %and a final lipid concentrations 25 mg/mL in buffer solution (AvantiPolar Lipids Inc-Alabaster, Ala.). 5.0 mg of selenocoxib-1-GSH will beadded to 1 milliliter of nanoliposome solution, the mixture dried undernitrogen gas and resuspended in 0.9% saline at room temperature to 60°C. Following rehydration, the material will be sonicated at 60° C. for30 minutes followed by extrusion at 60° C. through a 100-nmpolycarbonate membrane using Avanti Mini Extruder (Avanti Polar LipidsInc—Alabaster, Ala.). Analysis of particle size and charge ofnanoliposomes will be performed by using a Malvern Zetasizer Nano,Malvern Instruments, UK). Liposomes containing selenocoxib-1-GSHaccording to this protocol will be homogenously distributed in nanosizerange of 70-80 nm with a neutral surface charge. Determination ofencapsulation efficiency of selenocoxib-1-GSH into nanoliposomes will bebased on UV-visible spectrophotometry and drug loading calculatedthrough % of encapsulation efficiency (total drugs−free drugs/totaldrugs×10). Size exclusion chromatography and dialysis will be used toremoving free compound. Drug loading of greater or equal to 60% ispredicted.

Therapeutic universality of selenocoxib-1-GSH for killing cancer cellsis examined by treating pancreatic (MiaPaca-2), breast (MDA-MB-231),prostate (PC-3) or sarcoma (HT-1080) and colon (Caco2) cell lines withthe drug and establishing the IC50 for each cell line, Table III.Selenocoxib-1-GSH kills the various cancer cell lines at 2-3-fold lowerconcentrations than normal fibroblast cells.

TABLE III Cell lines Celecoxib Selenocoxib-1-GSH MDA-MB-231(Breast) >100 15.4 ± 2.51 PC-3 (Prostate) >100 16.4 ± 2.11 MiaPaca-2(Pancreas) >100 13.3 ± 1.01 HT-1080 (Fibro sarcoma) >100  9.1 ± 1.89Caco-2 (Colon) >100 16.1 ± 0.87

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A composition comprising a selenium-containing COX-2 inhibitor,selected from the group consisting of: selenocoxib-1-glutathione(selenocoxib-1-GSH), selenocoxib-1-cysteine,selenocoxib-1-N-aceylcysteine (selenocoxib-1-NAC) and a mixture of twoor more thereof.
 2. The composition of claim 1, wherein theselenium-containing COX-2 inhibitor is selenocoxib-1-glutathione(selenocoxib-1-GSH).
 3. The composition of claim 1, wherein theselenium-containing COX-2 inhibitor is selenocoxib-1-cysteine.
 4. Thecomposition of claim 1, wherein the selenium-containing COX-2 inhibitoris selenocoxib-1-N-aceylcysteine (selenocoxib-1-NAC).
 5. Apharmaceutical composition, comprising: a selenium-containing COX-2inhibitor selected from the group consisting of selenocoxib-1-GSH,selenocoxib-1-cysteine, selenocoxib-1-NAC; and a mixture of two or morethereof; and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5, wherein the selenium-containingCOX-2 inhibitor is selenocoxib-1-GSH.
 7. The pharmaceutical compositionof claim 5, wherein the pharmaceutical composition is formulated fortopical application.
 8. The pharmaceutical composition of claim 5,wherein the pharmaceutically acceptable carrier comprises a particulatecarrier.
 9. The pharmaceutical composition of claim 5, wherein thepharmaceutically acceptable carrier comprises a nanoparticulate carrier.10. The pharmaceutical composition of claim 5, comprising a plurality ofnanoliposomes, the nanoliposomes comprising selenocoxib-1-GSH,selenocoxib-1-cysteine, selenocoxib-1-NAC or a mixture of any two ormore thereof, the nanoliposomes having an average particle size in therange of 1-100 nm.
 11. The pharmaceutical composition of claim 5,further comprising an additional therapeutic agent.
 12. A method oftreating a subject, comprising: administering a therapeuticallyeffective amount of a pharmaceutical composition comprising aselenium-containing COX-2 inhibitor selected from the group consistingof: selenocoxib-1-GSH, selenocoxib-1-cysteine, selenocoxib-1-NAC and amixture of two or more thereof.
 13. The method of claim 12, wherein thepharmaceutical composition comprises a plurality of nanoliposomes, thenanoliposomes comprising selenocoxib-1-GSH, selenocoxib-1-cysteine,selenocoxib-1-NAC or any two or more thereof, the nanoliposomes havingan average particle size in the range of 1-100 nm.
 14. The method ofclaim 12, wherein the pharmaceutical composition is formulated fortopical application.
 15. The method of claim 12, wherein thepharmaceutical composition comprises a particulate carrier, ananoparticulate carrier and/or an additional therapeutic agent.
 16. Themethod of claim 12, wherein the subject is human.
 17. The method ofclaim 12, wherein the subject has or is suspected of having cancer. 18.The method of claim 12, wherein the subject has or is suspected ofhaving breast cancer, lung cancer, prostate cancer, colon cancer, livercancer or melanoma.
 19. The method of claim 12, wherein the subject hasor is suspected of having cancer characterized by increased COX-2protein or nucleic acid, increased COX-2 activity and/or increased Aktactivity compared to a control.
 20. The method of claim 17, whereinadministering the therapeutically effective amount of the pharmaceuticalcomposition to the subject detectably increases apoptosis and/ordecreases proliferation of cells of the cancer and has negligible effecton non-cancer cells.
 21. The method of claim 17, wherein administeringthe therapeutically effective amount of the pharmaceutical compositionto the subject detectably decreases free radicals and/or reactive oxygenspecies of cells of the cancer and has negligible effect on non-cancercells.
 22. The method of claim 17, further comprising: assaying of COX-2protein, COX-2 nucleic acid, COX-2 activity and/or Akt activity in cellsof the cancer prior to administration of the pharmaceutical compositionto determine the level of expression of COX-2 protein in the cells ofthe cancer compared to a control, the level of expression of COX-2nucleic acid in the cells of the cancer compared to a control, the levelof activity of COX-2 in the cells of the cancer compared to a controland/or the level of activity of Akt in the cells of the cancer comparedto a control.
 23. The method of claim 17, further comprising: assayingof COX-2 protein, COX-2 nucleic acid, COX-2 activity and/or Akt activityin cells of the cancer after administration of the pharmaceuticalcomposition to determine the level of expression of COX-2 protein in thecells of the cancer compared to a control, the level of expression ofCOX-2 nucleic acid in the cells of the cancer compared to a control, thelevel of activity of COX-2 in the cells of the cancer compared to acontrol and/or the level of activity of Akt in the cells of the cancercompared to a control.
 24. The method of claim 17, further comprising:assaying of COX-2 protein, COX-2 nucleic acid, COX-2 activity and/orAkt3 activity in cells of the cancer prior to administration of thepharmaceutical composition to determine the level of expression of COX-2protein in the cells of the cancer compared to a control, the level ofexpression of COX-2 nucleic acid in the cells of the cancer compared toa control, the level of activity of COX-2 in the cells of the cancercompared to a control and/or the level of activity of Akt3 in the cellsof the cancer compared to a control.
 25. The method of claim 17, furthercomprising: assaying of COX-2 protein, COX-2 nucleic acid, COX-2activity and/or Akt3 activity in cells of the cancer afteradministration of the pharmaceutical composition to determine the levelof expression of COX-2 protein in the cells of the cancer compared to acontrol, the level of expression of COX-2 nucleic acid in the cells ofthe cancer compared to a control, the level of activity of COX-2 in thecells of the cancer compared to a control and/or the level of activityof Akt3 in the cells of the cancer compared to a control, wherein thecontrol is a test sample obtained from the subject prior toadministration of the pharmaceutical composition.
 26. The method ofclaim 17, further comprising administration of an adjunct anti-cancertreatment to the subject.
 27. The method of claim 12 wherein thepharmaceutical composition is administered topically.